RESUMEN
Two strategies were aimed at identifying immunogenically optimized peptides for the potential use in the formulation of an effective prophylactic or therapeutic HIV-1 vaccine. Three CTL epitopes were investigated: Gag p24(19-27) TV9, Gag p17(77-85) SL9, and RT(309-317) IV9. The first strategy derives from the hypothesis that a number of rare mutant CTL epitopes of HIV-1 may be more immunogenic than the common ones. As such, these rare mutant sequences might be highly effective in generating cross reactive anti-HIV-1 CTL responses against a range of mutant sequences. As anticipated, several rare mutant peptide sequences were identified that generated strong CTL responses against both the consensus sequences and several naturally occurring mutants in human PBL cultures primed ex vivo and in HLA-A2 transgenic mice immunized in vivo. Finally, to reach beyond the sequence diversity of the "natural" library of mutated sequences, a synthetic combinatorial peptide library was screened with a TV9 specific T-cell line; this resulted in the identification of an immunogenically optimized mimic peptide sequence that provoked highly effective CTL immune responses against TV9 and mutants. Sequence homologies between the natural mutants and synthetic mimic may provide insight into key contact positions in the MHC/TCR/peptide complex.
Asunto(s)
Técnicas Químicas Combinatorias , Epítopos de Linfocito T/inmunología , VIH/inmunología , Mutación , Biblioteca de Péptidos , Péptidos/inmunología , Linfocitos T Citotóxicos/inmunología , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/fisiología , Animales , Fármacos Anti-VIH/inmunología , Fármacos Anti-VIH/metabolismo , Células Cultivadas , Reacciones Cruzadas/genética , Reacciones Cruzadas/inmunología , Pruebas Inmunológicas de Citotoxicidad , Epítopos de Linfocito T/genética , Epítopos de Linfocito T/metabolismo , VIH/genética , Proteína p24 del Núcleo del VIH/genética , Proteína p24 del Núcleo del VIH/metabolismo , Antígeno HLA-A2/genética , Humanos , Ratones , Ratones Transgénicos , Imitación Molecular , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Péptidos/genética , Péptidos/metabolismo , Linfocitos T Citotóxicos/metabolismo , Productos del Gen env del Virus de la Inmunodeficiencia Humana/genética , Productos del Gen env del Virus de la Inmunodeficiencia Humana/metabolismo , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
PURPOSE: Various lectins bind specifically to oligosaccharides on intestinal cells. Exploiting this specificity, Ulex europaeus agglutinin I (UEA1) has been used as a ligand for targeted oral vaccine delivery to M cells (antigen-presenting cells) in follicle-associated epithelium. In this study we characterized compounds identified from mixture-based positional scanning synthetic combinatorial libraries, which mimic UEA1 and, thus, may have properties applicable to targeted drug delivery. METHODS: Two UEA1 mimetics were synthesized and their activity was verified on live cells. The ability of the lead compound, a tetragalloyl D-Lysine amide construct (4-copy gallic acid construct), to deliver dye-loaded polystyrene particles to M cells was assessed in an in situ mouse gut loop model. RESULTS: The 4-copy gallic acid construct inhibited UEA1 binding to Caco-2 cell membranes with an IC50 of 3 microM, a 650- to 5000-fold increase over the natural UEA1 substrate alpha-L-fucose. The biotin-labeled derivative of this construct demonstrated comparable binding activity as verified on live cells by fluorescence-activated cell sorting. Preclinical studies confirmed its ability to mediate M cell-specific delivery of streptavidin-coated particles in vivo. CONCLUSIONS: Polyphenolic compounds, D-Lysine scaffolds with multiple galloyl groups, can mimic functional activities of UEA1. Properties of such molecules, including low molecular weight, stability, ease of synthesis and low cost, highlight their potential for application in targeted vaccine delivery.