RESUMEN
HIV-1 candidate vaccines expressing an artificial polyprotein comprising Gag, Pol and Nef (GPN) and a secreted envelope protein (Env) were shown in recent Phase I/II clinical trials to induce high levels of polyfunctional T cell responses; however, Env-specific responses clearly exceeded those against Gag. Here, we assess the impact of the GPN immunogen design and variations in the formulation and vaccination regimen of a combined GPN/Env DNA vaccine on the T cell responses against the various HIV proteins. Subtle modifications were introduced into the GPN gene to increase Gag expression, modify the expression ratio of Gag to PolNef and support budding of virus-like particles. I.m. administration of the various DNA constructs into BALB/c mice resulted in an up to 10-fold increase in Gag- and Pol-specific IFNγ(+) CD8(+) T cells compared to GPN. Co-administering Env with Gag or GPN derivatives largely abrogated Gag-specific responses. Alterations in the molar ratio of the DNA vaccines and spatially or temporally separated administration induced more balanced T cell responses. Whereas forced co-expression of Gag and Env from one plasmid induced predominantly Env-specific T cells responses, deletion of the only H-2(d) T cell epitope in Env allowed increased levels of Gag-specific T cells, suggesting competition at an epitope level. Our data demonstrate that the biochemical properties of an artificial polyprotein clearly influence the levels of antigen-specific T cells, and variations in formulation and schedule can overcome competition for the induction of these responses. These results are guiding the design of ongoing pre-clinical and clinical trials.
Asunto(s)
Productos del Gen gag/inmunología , VIH-1/inmunología , Vacunas de ADN , Productos del Gen env del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/inmunología , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/inmunología , Animales , Ensayos Clínicos Fase III como Asunto , Femenino , Productos del Gen gag/biosíntesis , Productos del Gen gag/genética , Células HEK293 , VIH-1/genética , Humanos , Ratones , Ratones Endogámicos BALB C , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas Sintéticas/administración & dosificación , Vacunas Sintéticas/genética , Vacunas Sintéticas/inmunología , Vacunas de Partículas Similares a Virus/inmunología , Productos del Gen env del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/biosíntesis , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
This study aimed to identify the characteristics of recombinant-adenovirus-modified PBMC-derived dendritic cells and their resistance to HIV-1 infection by integrating the CCR5∆32, CCR5siRNA, HIV-1 pol and HIV-1 int genes into a recombinant adenovirus vector using the AdEasy system. Dendritic cells (DCs) were isolated from human PBMCs from blood of healthy donors. The expression of CCR5∆32, CCR5, CXCR4 and HIV-1 p24 in PBMCs or modified cells was measured by western blot, p24 expression in cell lysates was measured by ELISA, and HIV-1 entry was measured by ß-galactosidase assay. Furthermore, T-cell immunity induced by the recombinant adenovirus was measured by ELISPOT assay. After the cells were modified by Ad-R5∆32siRNA, the expression of CCR5∆32 increased, while the expression of CCR5 and CXCR4 decreased. There was no adverse effect of adenoviral gene transfer on DC development. CD83 expression on the surface of mature DCs did not change after gene transfer. The expression of p24 remained at low levels in modified cells when challenged by HIV-1. The modified cells showed resistance to HIV-1 infection. Results indicated that recombinant-adenovirus-modified cells demonstrated good resistance to HIV-1 infection. Modification of HSC-derived immune cells, such as DCs, may be a potent strategy to resist HIV-1 infection.