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
The development of cervical cancer is associated with infection by oncogenic human papillomaviruses (HPVs), of which type 16 (HPV16) is the most prevalent in HPV-induced malignant diseases. The viral oncoproteins E6 and E7 are convenient targets for anti-tumor immunization. To adapt the corresponding genes for DNA vaccination, their oncogenicity needs to be reduced and immunogenicity enhanced. The main modifications for achieving these aims include mutagenesis, rearrangement of gene parts, and fusion with supportive cellular or viral/bacterial genes or their functional parts. As HPVs are strictly human specific, an animal model of HPV infection does not exist. Therefore, immunization against HPV-induced tumors is most frequently tested in mouse models utilizing transplantable syngeneic tumor cells producing the HPV16 E6/E7 oncoproteins. In this chapter, one such cell line designated TC-1 is characterized and the effect of immunization with the modified E7 fusion gene against TC-1-induced subcutaneous tumors is described. As down-regulation of MHC class I molecules is one of the most important escape mechanisms of cervical carcinoma cells, the TC-1/A9 clone with reversibly reduced MHC class I expression has been developed and, herein, its response to DNA vaccination is also shown and compared with that of the TC-1 cells.
- MeSH
- biolistika přístrojové vybavení MeSH
- DNA vakcíny aplikace a dávkování chemie genetika imunologie MeSH
- lidé MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- nádory děložního čípku patologie prevence a kontrola MeSH
- proliferace buněk MeSH
- protinádorové vakcíny aplikace a dávkování chemie genetika imunologie MeSH
- vakcinace přístrojové vybavení MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8(+) T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.
- MeSH
- adjuvancia imunologická aplikace a dávkování MeSH
- bcr-abl fúzní proteiny genetika imunologie metabolismus MeSH
- biolistika MeSH
- CD8-pozitivní T-lymfocyty účinky léků metabolismus patologie MeSH
- DNA vakcíny MeSH
- experimentální nádory imunologie patologie terapie MeSH
- imunita účinky léků MeSH
- levamisol aplikace a dávkování MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední BALB C MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- oligodeoxyribonukleotidy aplikace a dávkování MeSH
- Papillomavirus E7 - proteiny genetika imunologie metabolismus MeSH
- protinádorové vakcíny MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- léčba diabetu,
- MeSH
- aplikace bukální MeSH
- aplikace inhalační MeSH
- aplikace intranazální MeSH
- aplikace kožní MeSH
- diabetes mellitus farmakoterapie terapie MeSH
- injekce tryskové metody přístrojové vybavení využití MeSH
- inzulin aplikace a dávkování škodlivé účinky terapeutické užití MeSH
- iontoforéza metody využití MeSH
- lidé MeSH
- liposomy terapeutické užití MeSH
- nanočástice terapeutické užití MeSH
- ultrazvuk MeSH
- způsoby aplikace léků MeSH
- Check Tag
- lidé MeSH
- MeSH
- aplikace inhalační MeSH
- aplikace kožní MeSH
- injekce intradermální přístrojové vybavení MeSH
- injekce intramuskulární přístrojové vybavení MeSH
- injekce tryskové přístrojové vybavení MeSH
- lidé MeSH
- vakcinace metody trendy MeSH
- vakcíny aplikace a dávkování MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- přehledy MeSH
- MeSH
- antigeny nádorové imunologie MeSH
- biolistika MeSH
- DNA vakcíny imunologie MeSH
- financování organizované MeSH
- imunizace MeSH
- injekce subkutánní MeSH
- mutace MeSH
- myši inbrední C57BL MeSH
- nádorové buněčné linie MeSH
- nádory plic patologie MeSH
- onkogenní proteiny virové genetika imunologie MeSH
- plazmidy MeSH
- proteiny tepelného šoku HSC70 imunologie MeSH
- transformované buněčné linie MeSH
- virová transformace buněk MeSH
- zvířata MeSH
- Check Tag
- ženské pohlaví MeSH
- zvířata MeSH
Vaccine strategies for the treatment of human papillomavirus-induced cervical cancer are based mainly on the human papillomavirus 16 E7 (HPV16 E7) oncoprotein. The immunogenicity of the E7 gene has been enhanced by its fusion to many different genes. Here, we linked a short sequence coding for the E7 peptide (aa 44-60) containing immunodominant epitopes for B and T cells to the 3' end of the gene coding for the whole coat protein (CP) of the poty-virus, potato virus A (PVA), and its deleted form (CPdel) with a short C-terminal deletion of 5 amino acids (LGVKG). CP-E7 and CPdel-E7 fusion proteins, just like CP alone, spontaneously assembled into virus-like particles in both procaryotic and eucaryotic cells. The CP-E7 and CPdel-E7 fusion genes induced slightly stronger E7-specific cytotoxic T-lymphocyte responses than the whole E7 gene, although they were still lower than those elicited by the previously constructed fusion gene, Sig/E7GGG/LAMP-1. The E7- and CP-specific antibody responses were not detected in mice vaccinated with CP-E7 and CPdel-E7 fusion genes. The CP-E7 and CPdel-E7 fusion genes protected mice against the development of tumors induced by TC-1 cells producing the E7 antigen and were also effective in the therapeutic setting, i.e. when the vaccination was performed after tumor cell administration. Their antitumor effect was comparable to those of the whole E7 gene and Sig/E7GGG/LAMP-1 fusion gene. There was no relevant difference between immune responses elicited by CP-E7 and CPdel-E7 DNA vaccination.
- MeSH
- biolistika MeSH
- buněčné linie MeSH
- buňky NIH 3T3 MeSH
- časové faktory MeSH
- cytotoxické T-lymfocyty cytologie MeSH
- DNA vakcíny MeSH
- ELISA MeSH
- financování organizované MeSH
- genetická terapie metody MeSH
- hmotnostní spektrometrie MeSH
- lidé MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádorové buněčné linie MeSH
- onkogenní proteiny virové chemie MeSH
- peptidy chemie MeSH
- plazmidy metabolismus MeSH
- Potyvirus genetika MeSH
- protinádorové látky farmakologie MeSH
- protinádorové vakcíny MeSH
- rekombinantní fúzní proteiny chemie MeSH
- RNA chemie MeSH
- sekvence aminokyselin MeSH
- sekvenční homologie aminokyselin MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
- technika přenosu genů MeSH
- transmisní elektronová mikroskopie MeSH
- transplantace nádorů MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
