Since its discovery, DNA vaccination has become an effective strategy for the development of vaccines against cancer including cervical carcinoma (CC). The formation of CC is associated with human papillomavirus (HPV) infection. Viral E6 and E7 oncoproteins are suitable targets for therapeutic vaccination. To adapt the HPV16 E6 oncogene for DNA immunisation, we performed several modifications. First we fused the E6 gene with the 5' or 3'-terminus of the Escherichia coli beta-glucuronidase (GUS) gene and showed enhanced immunogenicity of the 3' fusion (GUS.E6). Then, as the E6 oncogene contains two alternative introns that result in the production of truncated forms of the E6 protein, we abolished the 5' splice site in the E6 gene. This modification completely eliminated the expression of the truncated E6 transcripts and thus increased the production of the full-length E6 protein. At the same time, it moderately reduced the immunogenicity of the modified non-fused (E6cc) or fused (GUS.E6cc) genes, probably as a consequence of the substitution in the immunodominant E6 epitope following the abolishment of the splice site. Furthermore, we reduced the oncogenicity of the E6 protein by two point mutations (E6GT) that, together, prevented E6-mediated p53 degradation. Finally, we constructed the GUS.E6GT gene characterized by enhanced safety and immunogenicity when compared with the wild-type E6 gene.
- MeSH
- bodová mutace MeSH
- buněčné linie MeSH
- cytokiny biosyntéza MeSH
- DNA vakcíny genetika imunologie MeSH
- exprese genu MeSH
- glukuronidasa genetika imunologie MeSH
- introny MeSH
- leukocyty mononukleární imunologie MeSH
- lidé MeSH
- lidský papilomavirus 16 genetika imunologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- nádory prevence a kontrola MeSH
- onkogenní proteiny virové genetika imunologie MeSH
- proteiny z Escherichia coli genetika imunologie MeSH
- protilátky virové krev MeSH
- rekombinantní fúzní proteiny genetika imunologie MeSH
- represorové proteiny genetika imunologie MeSH
- vakcíny proti papilomavirům genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Tattooing has been shown to be very efficient at inducing immunity by vaccination with DNA vaccines. In this study, we examined the usability of tattooing for delivery of peptide vaccines. We compared tattooing with subcutaneous (s.c.) needle injection using peptides derived from human papillomavirus type 16 (HPV16) proteins. We observed that higher peptide-specific immune responses were elicited after vaccination with the simple peptides (E7(44-62) and E7(49-57)) and keyhole limpet hemocyanin-(KLH)-conjugated peptides (E7(49-57), L2(18-38) and L2(108-120)) with a tattoo device compared to s.c. inoculation. The administration of the synthetic oligonucleotide containing immunostimulatory CpG motifs (ODN1826) enhanced the immune responses developed after s.c. injection of some peptides (E7(44-62), KLH-conjugated L2(18-38) and L2(108-120)) to levels close to or even comparable to those after tattoo delivery of identical peptides with ODN1826. The highest efficacy of tattooing was observed in combination with ODN1826 for the vaccination with the less immunogenic E6(48-57) peptide and KLH-conjugated and non-conjugated E7(49-57) peptides which form the visible aggregates that could negatively influence the development of immune responses after s.c. injection but probably not after tattooing. In summary, we first evidenced that tattoo administration of peptide vaccines that might be useful in some cases efficiently induced both humoral and cell-mediated immune responses.
- MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- DNA vakcíny imunologie MeSH
- hemokyanin imunologie MeSH
- lidský papilomavirus 16 imunologie MeSH
- molekulární sekvence - údaje MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- onkogenní proteiny virové imunologie MeSH
- Papillomavirus E7 - proteiny MeSH
- peptidové fragmenty MeSH
- represorové proteiny imunologie MeSH
- sekvence aminokyselin MeSH
- tetování MeSH
- vakcinace MeSH
- vakcíny proti papilomavirům imunologie MeSH
- virové plášťové proteiny imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
- Publikační typ
- abstrakt z konference MeSH
Therapeutic DNA vaccines against oncogenic infection with human papillomavirus type 16 (HPV16) are mostly targeted against viral oncoproteins E7 and E6. To adapt the E7 oncoprotein for DNA immunization, we have previously reduced its oncogenicity by modification of the Rb-binding site and enhanced immunogenicity of the modified E7GGG gene by the fusion with the 5'-terminus of the gene encoding E. coli beta-glucuronidase (GUS). In this study, we attempted to improve immunogenicity of the GUS-based anti-E7 vaccines by increasing the steady-state level of fusion proteins. We fused deletion mutants of E7GGG and codon-optimized E7GGG with the 5'-terminus of GUS and unaltered E7GGG with the 3'-terminus of GUS. Furthermore, we mutated the initiation codon of the GUS gene in the E7GGG.GUS construct, as GUS alone was produced from this fusion gene. We found that only the fusion of E7GGG with the 3'-terminus of GUS (GUS.E7GGG) and deletion mutants of E7GGG with the 5'-terminus of GUS increased the steady-state level of fusion proteins in transfected human 293T cells. Analysis of immune reactions induced in mice by vaccination via a gene gun showed that the increased steady-state level of fusion proteins resulted in augmented production of E7-specific antibodies, but did not enhance cell-mediated anti-tumor immunity. Finally, we joined the signal sequence of the adenoviral E3 protein with GUS.E7GGG. This modification led to the predominant localization of the fusion protein in the endoplasmic reticulum and enhancement of CD8+ T-cell response, while antibody production was reduced. In conclusion, we found modifications of the E7GGG.GUS fusion gene that augmented either humoral or cell-mediated immune responses.
- MeSH
- buňky NIH 3T3 MeSH
- cytotoxické T-lymfocyty imunologie MeSH
- DNA vakcíny imunologie MeSH
- financování organizované MeSH
- glukuronidasa genetika imunologie MeSH
- lidský papilomavirus 16 imunologie MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- onkogenní proteiny virové genetika imunologie MeSH
- Papillomavirus E7 - proteiny MeSH
- protilátky virové krev MeSH
- rekombinantní fúzní proteiny imunologie MeSH
- vakcíny proti papilomavirům imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- 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
- antitumorózní látky farmakologie 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 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
- MeSH
- DNA vakcíny terapeutické užití MeSH
- faktor stimulující granulocyto-makrofágové kolonie genetika MeSH
- finanční podpora výzkumu jako téma MeSH
- interleukin-2 genetika MeSH
- myši MeSH
- nádory terapie MeSH
- protinádorové vakcíny aplikace a dávkování terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- MeSH
- aktivní imunoterapie MeSH
- antigeny nádorové imunologie MeSH
- DNA vakcíny MeSH
- experimentální nádory prevence a kontrola MeSH
- finanční podpora výzkumu jako téma MeSH
- myši MeSH
- onkogenní proteiny virové MeSH
- Papillomaviridae imunologie MeSH
- protinádorové vakcíny terapeutické užití MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- MeSH
- cytokiny fyziologie MeSH
- DNA vakcíny genetika chemie imunologie MeSH
- fúze genů MeSH
- myši MeSH
- nádory děložního čípku genetika terapie virologie MeSH
- onkogenní proteiny virové fyziologie MeSH
- vazebná místa genetika imunologie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- srovnávací studie MeSH
