The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

• MeSH
• aminokyselinové motivy genetika   MeSH
• biolistika MeSH
• buňky NIH 3T3 MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• cysteinové endopeptidasy genetika   metabolismus   MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• experimentální nádory imunologie   terapie   MeSH
• HEK293 buňky MeSH
• imunoterapie metody   MeSH
• interferon gama metabolismus   MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• peptidové fragmenty genetika   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• T-lymfocyty pomocné-indukující imunologie   MeSH
• tetanový toxin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

In DNA vaccination, CD4(+) T-cell help can be enhanced by fusion of a gene encoding an immunization protein with a foreign gene or its part providing T(h) epitopes. To study the effect of helper epitope localization in a protein molecule, the influence of the vicinity of the helper epitope, and the impact of chimeric protein cellular localization, we fused the helper epitope p30 from tetanus toxin (TT, aa 947-967) with the N- or C-terminus of the mutated E7 oncoprotein (E7GGG) of human papillomavirus type 16, enlarged the p30 epitope with the flanking residues containing potential protease-sensitive sites and altered the cellular localization of the fusion constructs by signal sequences. The p30 epitope enhanced the E7-specific response, but only in constructs without added signal sequences. After localization of the fusion proteins into the endoplasmic reticulum and endo/lysosomal compartment, the TT-specific T(h)2 response was increased. The synthetic Pan DR epitope (PADRE) induced a stronger E7-specific response than the p30 epitope and its stimulatory effect was not limited to nuclear/cytoplasmic localization of the E7 antigen. These results suggest that in the optimization of immune responses by adding helper epitopes to DNA vaccines delivered by the gene gun, the cellular localization of the antigen needs to be taken into account.

• MeSH
• biolistika metody   MeSH
• buňky NIH 3T3 MeSH
• cytokiny metabolismus   MeSH
• DNA vakcíny aplikace a dávkování   farmakologie   MeSH
• endoplazmatické retikulum imunologie   metabolismus   MeSH
• HEK293 buňky MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• Papillomavirus E7 - proteiny genetika   metabolismus   farmakologie   MeSH
• peptidové fragmenty genetika   farmakologie   MeSH
• plazmidy aplikace a dávkování   MeSH
• rekombinantní fúzní proteiny metabolismus   farmakologie   MeSH
• tetanový toxin genetika   farmakologie   MeSH
• vakcína proti malárii farmakologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

• MeSH
• botulotoxiny genetika   chemie   toxicita   MeSH
• Clostridium genetika   metabolismus   MeSH
• extrachromozomální dědičnost MeSH
• lidé MeSH
• lyzogenie MeSH
• nervový systém účinky léků   MeSH
• paralýza chemicky indukované   MeSH
• tetanový toxin genetika   chemie   toxicita   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• přehledy MeSH