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  • 1
    Keywords: PEPTIDE ; CELLS ; EXPRESSION ; IN-VITRO ; tumor ; TUMOR-CELLS ; Germany ; human ; PROTEIN ; PROTEINS ; TUMORS ; MICE ; RELEASE ; COMPLEX ; RESPONSES ; COMPLEXES ; SERA ; REDUCTION ; DENDRITIC CELLS ; BINDING ; SEQUENCE ; antibodies ; NEUTRALIZING ANTIBODIES ; PARTICLES ; ASSAY ; ESCHERICHIA-COLI ; GLUTATHIONE ; LYMPHOCYTES ; VIRUS-LIKE PARTICLES ; HPV ; HPV16 ; VACCINE ; IMMUNE-RESPONSE ; vaccination ; L1 ; SEQUENCE-ANALYSIS ; glutathione-S-transferase ; HPV PSEUDOVIRIONS ; NASAL IMMUNIZATION
    Abstract: We analyzed capsomeres of human papillomavirus type 16 (HPV16) consisting of the L1 major structural protein for their ability to trigger a cytotoxic T-cell (CTL) response. To this end, we immunized C57BL/6 mice and used the L1(165-173) peptide for ex vivo restimulation of splenocytes prior to analysis (Cr-51 release assay and enzyme-linked immunospot assay [ELISPOT]). This peptide was identified in this study as a D-b-restricted naturally processed CTL epitope by HPV16 L1 sequence analysis, major histocompatibility complex class I binding, and Cr-51 release assays following immunization of C57BL/6 mice with HPV16 L1 virus-like particles (VLPs). HPV16 L1 capsomeres were obtained by purification of HPV16 L1 lacking 10 N-terminal amino acids after expression in Escherichia coli as a glutathione S-transferase fusion protein (GST-HPV16 L1DeltaN10). Sedimentation analysis revealed that the majority of the purified protein consisted of pentameric capsomeres, and assembled particles were not observed in minor contaminating higher-molecular-weight material. Subcutaneous (s.c.) as well as intranasal immunization of C57BL/6 m ice with HPV16 L1 capsomeres triggered an L1-specific CTL response in a dose- dependent manner as measured by ELISPOT and Cr-51 release as say. Significant reduction of contaminating bacterial endotoxin (lipopolysaccharide) from the capsomere preparation did not diminish the immunogenicity. Antibody responses (serum and vaginal) were less robust under the experimental conditions employed. In addition, s.c. vaccination with HPV16 L1 capsomeres induced regression of established tumors expressing L1 determinants (C3 tumor cells). Our data demonstrate that capsomeres are potent inducers of CTL responses similar to completely assembled T=7 VLPs. This result is of potential relevance for the development of (combined prophylactic and therapeutic) HPV-specific vaccines, since capsomeres can be produced easily and also can be modified to incorporate heterologous sequences such as early HPV proteins
    Type of Publication: Journal article published
    PubMed ID: 12663770
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  • 2
    Keywords: CANCER ; CELLS ; IN-VITRO ; tumor ; Germany ; human ; IN-VIVO ; THERAPY ; VITRO ; SYSTEM ; DISEASE ; RISK ; GENE ; GENE-TRANSFER ; DNA ; INFECTION ; INDUCTION ; ANTIGEN ; DENDRITIC CELLS ; T cells ; T-CELLS ; E7 ; OPEN READING FRAME ; papillomavirus ; HUMANS ; ASSAY ; WOMEN ; cervical cancer ; CERVICAL-CANCER ; human papillomavirus ; HIGH-RISK ; ONCOGENE ; TRANSFORMATION ; HUMAN-PAPILLOMAVIRUS ; VACCINE ; SAFETY ; EPITOPE ; IMMUNOTHERAPY ; PLASMID DNA ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; IMMUNIZATION ; MUTATIONAL ANALYSIS ; ASSAYS ; cytotoxic T lymphocyte ; BACTERIAL-DNA ; RISK-FACTOR ; in vivo ; tumor regression ; tumor protection ; ENHANCED IMMUNOGENICITY ; gene shuffling ; HPV16 E7 ; in vitro immunization
    Abstract: Anew and very promising approach in vaccine development is the application of naked DNA. In comparison to conventional vaccines it offers several advantages, especially if there is a need for the development of low cost vaccines. Infection with high-risk human papillomaviruses (hr-HPVs) is the major risk factor for the development of cervical cancer (cc), the third most common cancer in women worldwide. The HPV E7 oncogene is constitutively expressed in HPV-infected cells and represents an excellent target for immune therapy of HPV-related disease. Therefore, we chose the HPV-16 E7 as model antigen in the development of a therapeutic DNA vaccine candidate. For safety reasons the use of a transforming gene like the HPV-16 E7 for DNA vaccination is not feasible in humans. In consequence we have generated an artificial ("shuffled") HPV-16 E7-gene (HPV-16 E7SH), containing all putative cytotoxic T-lymphocyte (CTLs) epitopes and exhibiting high safety features. Here, we show the induction of a strong E7-wildtype (E7WT) directed cellular and humoral immune response including tumor protection and regression after in Vivo immunization in the murine system. Moreover, the vaccine candidate demonstrated immunogenicity in humans, demonstrated by priming of antigen-specific T cells in vitro. Importantly, the artificial HPV-gene has completely lost its transforming properties as measured in soft agar transformation assays. These results may be of importance for the development of vaccines based on oncogenes or oncoproteins. (c) 2006 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16472545
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  • 3
    Keywords: CANCER ; CELLS ; radiotherapy ; tumor ; CELL ; COMBINATION ; Germany ; human ; IN-VIVO ; SUPPORT ; GENE ; GENES ; PROTEIN ; SURGERY ; MICE ; TIME ; PATIENT ; RESPONSES ; DNA ; IFN-GAMMA ; INDUCTION ; ANTIGEN ; ANTIGENS ; T cells ; T-CELL ; T-CELLS ; cytokines ; IMMUNE-RESPONSES ; TARGET ; virus ; HUMANS ; cervical cancer ; CERVICAL-CANCER ; chemotherapy ; DELIVERY ; HPV ; HUMAN-PAPILLOMAVIRUS ; VACCINE ; GRANZYME-B ; ANTIGEN-PRESENTING CELLS ; immune response ; IMMUNE-RESPONSE ; IMMUNOTHERAPY ; IMMUNOGENICITY ; human papilloma virus ; ADJUVANT ; PLASMID DNA ; CYTOTOXIC T-LYMPHOCYTES ; IMMUNIZATION ; CYTOKINE ; REGRESSION ; secretion ; LIFE ; ENHANCEMENT ; cancer vaccine ; QUALITY-OF-LIFE ; DNA vaccine ; quality of life ; USA ; immunology ; tumor regression ; gene shuffling ; virology ; 3 ; Genetic ; therapeutic ; TUMOR-REGRESSION ; EXPRESSION CASSETTES ; genetic adjuvants ; gynecology
    Abstract: Treatment of patients with cervical cancer by conventional methods (mainly surgery, but also radiotherapy and chemotherapy) results in a significant loss in quality of life. A therapeutic DNA vaccine directed to tumor-specific antigens of the human papilloma virus (HPV) could be an attractive treatment option. We have developed a nontransforming HIV-16 E7-based DNA vaccine containing all putative T cell epitopes (HPV-16 E7SH). DNA vaccines, however, are less immunogenic than protein- or peptide-based vaccines in larger animals and humans. In this study, we have investigated an adjuvant gene support of the HPV-16 E7SH therapeutic cervical cancer vaccine. DNA encoded cytokines (IL-2, IL-12, GM-CSF, IFN-gamma) and the chemokine M1P1-alpha were coapplied either simultaneously or at different time points pre- or post-E7SH vaccination. In addition, sequence-optimized adjuvant genes were compared to wild type genes. Three combinations investigated lead to an enhanced iFN-gamma response of the induced T cells in mice. Interestingly, IFN-1 secretion of splenocytes did not strictly correlate with tumor response in tumor regression experiments. Gene-encoded Mill-la applied 5 days prior to E7SH-immunization combined with IFN-gamma or 1L-12 (3 days) or IL-2 (5 days) post immunization lead to a significantly enhanced tumor response that was clearly associated with granzyme B secretion and target cells lysis. Our results suggest that a conditioning application and combination with adjuvant genes may be a promising strategy to enhance synergistically immune responses by DNA immunization for the treatment of cervical cancer. (C) 2009 UICC
    Type of Publication: Journal article published
    PubMed ID: 19358269
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  • 4
    Keywords: OPTIMIZATION ; CANCER ; CELLS ; EXPRESSION ; tumor ; Germany ; GENE ; GENES ; PROTEIN ; EFFICIENCY ; TUMORS ; MICE ; DNA ; INDUCTION ; ANTIGEN ; T-CELL ; T-CELLS ; E7 ; papillomavirus ; SEQUENCE ; CODON USAGE ; human papillomavirus ; TYPE-16 ; HPV ; MAMMALIAN-CELLS ; VACCINE ; HUMAN-PAPILLOMAVIRUS TYPE-16 ; IMMUNOGENICITY ; CTL ; PLASMODIUM-FALCIPARUM ; T-cell response ; TRANSFECTION ; TRANSLATION ; ENHANCEMENT ; CODON ; DNA VACCINE POTENCY ; HPV 16 E7 expression clones
    Abstract: Immunization with a codon-optimized HPV 16 E7 gene was shown to yield higher levels of U-specific cytotoxic T cells [Liu WJ, Gao F, Zhao KN, Zhao W. Fernando GJ, Thomas R. et al. Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity. Virology 2002;301:43]. Here, we sought to verify the hypothesis that there is a direct correlation between the level of protein expression and immunogenicity in mice. We generated HPV 16 E7 expression plastmids where the genes were inserted either as authentic sequence (wt) or after optimizing the codons for use in mammalian cells (opt). For enhancement of translation of the E7 gene a 5' Kozak sequence (K) was added. Transfection experiments revealed the strength of expression in the order of E7opt + K, E7opt - K, E7wt + K and E7wt - K. After immunization of C57/136 mice we observed an equally strong CD8 divided by T-cell response with the E7opt plasmids (+ or -K), followed by the E7wt + K and E7wt - K DNAs. The same difference in efficiency obtained in tumor protection experiments. Regression of pre-existing tumors and CTL activity was observed only with the E7opt divided by K plasmid. From these data we conclude that the level of protein expression correlates with the efficiency of CTL response and hence testing by transfection of cells in culture may allow a pre-selection of expression plasmids prior to DNA immunization. (C) 2004 Elsevier Ltd. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 15629358
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