An effective HIV vaccine will likely need to induce potent and broad-based immunity, including CD8+ T cell responses. Hence, a quantitative assay to measure such responses in animal models will be important in the evaluation of candidate HIV vaccines. We show here that a single immunization with HIV DNA vaccines, followed by challenge with recombinant vaccinia virus expressing the relevant HIV antigen, allows quantitative assessment of CD8+ T cell responses. These responses can be profound (>30% of total CD8+ T cells) and directly reflect the level of memory CD8+ T cells at the time of challenge. Therefore, this assay will facilitate the selection of promising HIV vaccine candidates for further evaluation.

ABSTRACTThe effectiveness of cationic microparticles with adsorbed DNA at inducing immune responses was investigated in mice, guinea pigs, and rhesus macaques. Plasmid DNA vaccines encoding human immunodeficiency virus (HIV) Gag and Env adsorbed onto the surface of cationic poly(lactide-coglycolide) (PLG) microparticles were shown to be substantially more potent than corresponding naked DNA vaccines. In mice immunized with HIVgagDNA, adsorption onto PLG increased CD8+T-cell and antibody responses by ∼100- and ∼1,000-fold, respectively. In guinea pigs immunized with HIVenvDNA adsorbed onto PLG, antibody responses showed a more rapid onset and achieved markedly higher enzyme-linked immunosorbent assay and neutralizing titers than in animals immunized with naked DNA. Further enhancement of antibody responses was observed in animals vaccinated with PLG/DNA microparticles formulated with aluminum phosphate. The magnitude of anti-Env antibody responses induced by PLG/DNA particles was equivalent to that induced by recombinant gp120 protein formulated with a strong adjuvant, MF-59. In guinea pigs immunized with a combination vaccine containing HIVenvand HIVgagDNA plasmids on PLG microparticles, substantially superior antibody responses were induced against both components, as measured by onset, duration, and titer. Furthermore, PLG formulation overcame an apparent hyporesponsiveness of theenvDNA component in the combination vaccine. Finally, preliminary data in rhesus macaques demonstrated a substantial enhancement of immune responses afforded by PLG/DNA. Therefore, formulation of DNA vaccines by adsorption onto PLG microparticles is a powerful means of increasing vaccine potency.

ABSTRACTThe ability to target antigen-presenting cells with vectors encoding desired antigens holds the promise of potent prophylactic and therapeutic vaccines for infectious diseases and cancer. Toward this goal, we derived variants of the prototype alphavirus, Sindbis virus (SIN), with differential abilities to infect human dendritic cells. Cloning and sequencing of the SIN variant genomes revealed that the genetic determinant for human dendritic cell (DC) tropism mapped to a single amino acid substitution at residue 160 of the envelope glycoprotein E2. Packaging of SIN replicon vectors with the E2 glycoprotein from a DC-tropic variant conferred a similar ability to efficiently infect immature human DC, whereupon those DC were observed to undergo rapid activation and maturation. The SIN replicon particles infected skin-resident mouse DC in vivo, which subsequently migrated to the draining lymph nodes and upregulated cell surface expression of major histocompatibility complex and costimulatory molecules. Furthermore, SIN replicon particles encoding human immunodeficiency virus type 1 p55Gagelicited robust Gag-specific T-cell responses in vitro and in vivo, demonstrating that infected DC maintained their ability to process and present replicon-encoded antigen. Interestingly, human and mouse DC were differentially infected by selected SIN variants, suggesting differences in receptor expression between human and murine DC. Taken together, these data illustrate the tremendous potential of using a directed approach in generating alphavirus vaccine vectors that target and activate antigen-presenting cells, resulting in robust antigen-specific immune responses.

ABSTRACT A major challenge for the next generation of human immunodeficiency virus (HIV) vaccines is the induction of potent, broad, and durable cellular immune responses. The structural protein Gag is highly conserved among the HIV type 1 (HIV-1) gene products and is believed to be an important target for the host cell-mediated immune control of the virus during natural infection. Expression of Gag proteins for vaccines has been hampered by the fact that its expression is dependent on the HIV Rev protein and the Rev-responsive element, the latter located on the env transcript. Moreover, the HIV genome employs suboptimal codon usage, which further contributes to the low expression efficiency of viral proteins. In order to achieve high-level Rev-independent expression of the Gag protein, the sequences encoding HIV-1 SF2 p55 Gag were modified extensively. First, the viral codons were changed to conform to the codon usage of highly expressed human genes, and second, the residual inhibitory sequences were removed. The resulting modified gag gene showed increases in p55 Gag protein expression to levels that ranged from 322- to 966-fold greater than that for the native gene after transient expression of 293 cells. Additional constructs that contained the modified gag in combination with modified protease coding sequences were made, and these showed high-level Rev-independent expression of p55 Gag and its cleavage products. Density gradient analysis and electron microscopy further demonstrated that the modified gag and gagprotease genes efficiently expressed particles with the density and morphology expected for HIV virus-like particles. Mice immunized with DNA plasmids containing the modified gag showed Gag-specific antibody and CD8 + cytotoxic T-lymphocyte (CTL) responses that were inducible at doses of input DNA 100-fold lower than those associated with plasmids containing the native gag gene. Most importantly, four of four rhesus monkeys that received two or three immunizations with modified gag plasmid DNA demonstrated substantial Gag-specific CTL responses. These results highlight the useful application of modified gag expression cassettes for increasing the potency of DNA and other gene delivery vaccine approaches against HIV.

Intramuscular injection of DNA vaccines elicits potent humoral and cellular immune responses in mice. However, DNA vaccines are less efficient in larger animal models and humans. To gain a better understanding of the factors limiting the efficacy of DNA vaccines, we used fluorescence-labeled plasmid DNA in mice to 1) define the macroscopic and microscopic distribution of DNA after injection into the tibialis anterior muscle, 2) characterize cellular uptake and expression of DNA in muscle and draining lymph nodes, and 3) determine the effect of modifying DNA distribution and cellular uptake by volume changes or electroporation on the magnitude of the immune response. Injection of a standard 50-μl dose resulted in the rapid dispersion of labeled DNA throughout the muscle. DNA was internalized within 5 min by muscle cells near the injection site and over several hours by cells that were located along muscle fibers and in the draining lymph nodes. Histochemical staining and analysis of mRNA expression in isolated cells by RT-PCR showed that the transgene was detectably expressed only by muscle cells, despite substantial DNA uptake by non-muscle cells. Reduction of the injection volume to 5 μl resulted in substantially less uptake and expression of DNA by muscle cells, and correspondingly lower immune responses against the transgene product. However, expression and immunogenicity were restored when the 5-μl injection was followed by electroporation in vivo. These findings indicate that distribution and cellular uptake significantly affect the immunogenicity of DNA vaccines.

DNA vaccines can prime broad-based immune responses in small animal models. In the present study, we sought to evaluate the relative ability of DNA vaccines to induce humoral and cellular immune responses. Using a DNA vaccine encoding HIV gag in mice, we observed that CD8+ T cell responses were primed more readily than were antibody responses, particularly at low doses of DNA. These CD8+ T cell responses were detected in spleen cells, as well as at local sites such as the lung and draining lymph nodes. The potency of the HIV gag DNA vaccine used was sufficient to prime strong CTL responses in macaques, but only low to undetectable antibody responses. Therefore, DNA vaccines appear able to prime strong, broad CTL but only modest antibody responses. These results may have implications on the development of vaccines against infectious diseases where both CTL and antibody responses are desired, such as HIV.