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  • 1
    Keywords: PEPTIDE ; CELLS ; EXPRESSION ; IN-VITRO ; BLOOD ; COMBINATION ; Germany ; IN-VIVO ; VITRO ; GENERATION ; TOOL ; PROTEIN ; DIFFERENTIATION ; LINES ; MICE ; PATIENT ; RESPONSES ; ANTIGEN ; SKIN ; T-CELL ; T-CELLS ; BINDING ; CELL-LINES ; RECOGNITION ; TRANSGENIC MICE ; DESIGN ; NUMBER ; LINE ; MELANOMA ; LYMPHOCYTES ; LIGANDS ; STRATEGIES ; EPITOPES ; IMMUNITY ; IMMUNOTHERAPY ; vaccination ; SELECTION ; NY-ESO-1 ; IMMUNOGENICITY ; PERIPHERAL-BLOOD ; cell lines ; T-cell response ; AUTOIMMUNITY ; RE ; monitoring ; tumor-antigen
    Abstract: Purpose: The frequently expressed differentiation antigen tyrosinase-related protein-2 (TRP-2) has repeatedly been described as a target of spontaneous cytotoxic T-cell responses in melanoma patients, suggesting that it might be an ideal candidate antigen for T cell -〉 based immunotherapy. As a prerequisite for immunization, T-cell epitopes have to be identified. Whereas a number of HLA class I-presented TRP-2-derived epitopes are known, information about HILA class 11 presented antigenic ligands recognized by CD4(+) T helper (Th) cells is limited. Experimental Design: The search for TRP-2-derived Th epitopes was carried out by competitive in vitro peptide binding studies with predicted HLA-DRB1 *0301 ligands in combination with peptide and protein immunizations of HLA-DRB1 *0301 transgenic mice. In vivo selected candidate epitopes were subsequently verified for their immunogenicity in human T-cell cultures. Results: This strategy led to the characterization of TRP-2(60-74) as an HLA-DRB1 *0301-restricted Th epitope. Importantly, TRP-2(60-74)- reactive human CD4+ Th cell lines, specifically recognizing target cells loaded with recombinant TRP-2 protein, could be established by repeated peptide stimulation of peripheral blood lymphocytes from several HLA-DRB1 *03(+) melanoma patients. Even short-term peptide stimulation of patients' peripheral blood lymphocytes showed the presence of TRP-260-74- reactive T cells, suggesting that these T cells were already activated in vivo. Conclusion: Peptide TRP-2(60-74) might be a useful tool for the improvement of immunotherapy and immune monitoring of melanoma patients
    Type of Publication: Journal article published
    PubMed ID: 16033842
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  • 2
    Keywords: PEPTIDE ; CELLS ; EXPRESSION ; IN-VITRO ; tumor ; TUMOR-CELLS ; BLOOD ; Germany ; IN-VIVO ; VITRO ; POPULATION ; TUMORS ; LINES ; PATIENT ; LIGAND ; RESPONSES ; IFN-GAMMA ; ANTIGEN ; DENDRITIC CELLS ; SKIN ; T-CELL ; T-CELLS ; RECOGNITION ; IDENTIFICATION ; LINE ; MELANOMA ; LYMPHOCYTES ; REGION ; LIGANDS ; EPITOPES ; IMMUNOTHERAPY ; vaccination ; PERIPHERAL-BLOOD ; RE ; MELANOMA-CELLS ; TYROSINASE-RELATED PROTEIN-2 ; dendritic cell ; T cell epitope ; T-CELL EPITOPE ; VACCINIA VIRUS ANKARA ; tumor antigen ; HOST-RANGE SELECTION ; TRP-2
    Abstract: Tyrosinase-related protein-2 (TRP-2) is a known target antigen of spontaneous cytotoxic T cell responses in melanoma patients. Its frequent expression in metastatic tumors suggests that it might be an ideal candidate antigen for T cell-based immunotherapy. To provide knowledge about TRP-2-derived T cell epitopes useful for immunotherapy we applied a "reverse immunology strategy" based on repeated in vitro peptide stimulation of peripheral blood lymphocytes (PBL) from normal donors with predicted HLA-A*01 ligands. This led to the identification of TRP-2(181-190) as the first HLA-A*01-presented TRP-2-derived epitope. T-cell lines specific for peptide TRP-2(181-190) could be established from PBL of 50% of the normal HLA-A*01(+) donors tested. Such T cells responded specifically to autologous dendritic cells transduced virally with TRP-2, as well as to HLA-A*01(+), TRP-2(+) melanoma cells, although tumor cells had to be pretreated with IFN-gamma to become susceptible to T cell recognition. Interestingly, short-term in vitro peptide stimulation of PBL from HLA-A*01(+) melanoma patients showed the presence of TRP-2(181-190)-reactive CD8(+) T cells in some donors, suggesting their in vivo sensitization. Because TRP-2(181-190) overlaps with the known HLA-A*0201-presented epitope TRP-2(180-188), an 11mer peptide encompassing both epitopes might be of specific value for vaccination of a broad population of melanoma patients. (C) 2005 Wiley-Liss, Inc
    Type of Publication: Journal article published
    PubMed ID: 15856458
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  • 3
    Keywords: PEPTIDE ; CELLS ; BLOOD ; CELL ; Germany ; human ; VIVO ; PROTEIN ; PROTEINS ; MICE ; RESPONSES ; INFECTION ; FAMILY ; DONOR ; T cells ; T-CELLS ; MEMBER ; MEMBERS ; MEMORY ; TRANSGENIC MICE ; IDENTIFICATION ; CELL-LINE ; LINE ; LYMPHOCYTES ; PEPTIDES ; EPITOPE ; HUMAN DENDRITIC CELLS ; FAMILIES ; ESCAPE ; VIRULENCE ; T helper cells ; BACTERIA ; spleen ; immune responses ; bacterial infection ; GROUP-A STREPTOCOCCUS ; LISTERIA-MONOCYTOGENES ; PNEUMOLYSIN
    Abstract: Cholesterol-binding cytolysins constitute an evolutionarily conserved family of pore-forming proteins expressed by different Gram-positive pathogens. Listeriolysin O, one well-characterized member of the cytolysin family, is also known to induce specific CD4 and CD8 T cell responses upon infection of mice with Listeria monocytogenes. Here we describe an HLA-DRB1*0301-restricted listeriolysin O-derived T cell epitope that is conserved among several members of the cytolysin family. An HLA-DRB1*0301-restricted CD4+T cell line, established from spleen lymphocytes of L. monocytogenes-infected HLA-DRB1*0301-transgenic mice, cross-reacted with a homologous peptide from perfringolysin O, a cytolysin expressed by Clostridium peifringens. Ex vivo analysis of infected mice revealed an even broader cross-reaction of T cells with homologous peptides derived from perfringolysin O, streptolysin O, and cereolysin O. Interestingly, a cross-reactive memory CD4+T cell response against the homologous peptides derived from listeriolysin O and perfringolysin O could also be detected in the blood from healthy HLA-DRB1*0301(+) human donors. Remarkably, this response was even present in donors who did not exhibit a memory T cell reactivity against a second, non-conserved HLA-DRB1*0301-restricted LLO-derived CD4 T cell epitope, suggesting that cytolysin-producing bacteria other than L. monocytogenes can stimulate a cross-reactive cytolysin-specific immunity. (c) 2006 Elsevier SAS. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16798043
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  • 4
    Keywords: PEPTIDE ; CANCER ; CANCER CELLS ; CELLS ; EXPRESSION ; tumor ; CELL ; Germany ; human ; INHIBITION ; MODEL ; GENE ; PROTEIN ; PROTEINS ; MONOCLONAL-ANTIBODY ; LINES ; MICE ; DNA ; INDUCTION ; ANTIGEN ; CELL-LINES ; E7 ; papillomavirus ; treatment ; SUSCEPTIBILITY ; antibodies ; antibody ; MOUSE ; DESIGN ; CERVICAL-CANCER ; CELL-LINE ; LINE ; CODON USAGE ; human papillomavirus ; TYPE-16 ; CANCER-CELLS ; HPV ; antigen presentation ; PEPTIDES ; E6 ; MONOCLONAL-ANTIBODIES ; HUMAN-PAPILLOMAVIRUS ; DEGRADATION ; VACCINE ; EPITOPE ; IMMUNE-RESPONSE ; RECOMBINANT VACCINIA ; HUMAN-PAPILLOMAVIRUS TYPE-16 ; MOUSE MODEL ; TARGETS ; cell lines ; CLASS-I MOLECULES ; IMMUNIZATION ; TRANSFECTION ; LEVEL ; PROTEIN-A ; tumor-antigen ; monoclonal antibodies ; function ; cytotoxic T lymphocyte ; E6 and E7 ; RELEVANCE ; PLASMID ; PRECURSOR ; E6 PROTEIN ; DNA immunization ; E7 PROTEIN ; CTL epitope ; CYTOTOXIC T-CELLS ; HPV 16 E6 ; RNA splicing ; THERAPEUTIC VACCINATION
    Abstract: The early proteins E6 and E7 of the cancer-related human papillomavirus type 16 (HPV 16) are constitutively expressed in cancer cells thus are targets for immune therapeutic approaches. Whereas previous studies have mainly focussed on the immunogenicity of E7 protein little is known about E6. In order to evaluate E6-specific DNA immunization strategies in a preclinical mouse model C57BL/6 mice were injected with plasmid pTHampE6 and analyzed for E6-specific CTL induction. CTL specific for the H2-K-b-restricted E6-derived epitope E6 48-57, were readily detectable among splenocytes of immunized animals, however, these CTL showed a differential recognition pattern on various E6-expressing target cells. Using a newly generated E6-specific monoclonal antibody we found that most cell lines expressing E6 encoded by the natural gene showed undetectable protein amounts and were ignored by E6-specific CTL. However, transfection of a codon optimized version of the E6 gene (E6opt) strongly enhanced protein expression levels within these cells turning them into susceptible target cells. Surprisingly, we found that E6-positive TC-1 cells, although recognized by E6-specific CTL, were totally devoid of any detectable E6 protein. Inhibition of proteasomal function by lactacystin treatment diminished E6-specific CTL recognition of TC-1 cells and RMA/E6opt transfectants accompanied by intracellular accumulation of E6 protein as observed in RMA/E6opt transfectants, but not in TC-1 cells. These data suggest that in TC-1 cells rapid degradation processes might prevent stable expression of E6 protein yet generate precursor peptides in amounts sufficient for MHC class I restricted antigen presentation. Thus, the results presented in this paper show that: (i) use of optimized codons in transfection experiments can improve susceptibility of target cells to E6-specific CTL recognition and (ii) lack of detectable protein within a cell does not necessarily indicate the absence of epitope presentation. Both findings are of potential relevance for the design of tumor vaccines. (c) 2006 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 16949679
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  • 5
    Keywords: TUMOR-CELLS ; carcinoma ; BREAST-CANCER ; chemotherapy ; LYMPHOCYTES ; IMMUNOTHERAPY ; immune cells ; DIFFERENTIATION ANTIGEN ; NY-BR-1 PROTEIN EXPRESSION ; ESTABLISHED MELANOMA
    Abstract: Breast cancer represents the second most common cancer type worldwide and has remained the leading cause of cancer related deaths among women. The differentiation antigen NY-BR-1 appears overexpressed in invasive mammary carcinomas compared to healthy breast tissue, thus representing a promising target antigen for T cell based tumor immunotherapy approaches. Since efficient immune attack of tumors depends on the activity of tumor antigen-specific CD4+ effector T cells, NY-BR-1 was screened for the presence of HLA-restricted CD4+ T cell epitopes that could be included in immunological treatment approaches. Upon NY-BR-1-specific DNA immunization of HLA-transgenic mice and functional ex vivo analysis, a panel of NY-BR-1-derived library peptides was determined that specifically stimulated IFNgamma secretion among splenocytes of immunized mice. Following in silico analyses, four candidate epitopes were determined which were successfully used for peptide immunization to establish NY-BR-1-specific, HLA-DRB1*0301- or HLA-DRB1*0401-restricted CD4+ T cell lines from splenocytes of peptide immunized HLA-transgenic mice. Notably, all four CD4+ T cell lines recognized human HLA-DR-matched dendritic cells (DC) pulsed with lysates of NY-BR-1 expressing human tumor cells, demonstrating natural processing of these epitopes also within the human system. Finally, CD4+ T cells specific for all four CD4+ T cell epitopes were detectable among PBMC of breast cancer patients, showing that CD4+ T cell responses against the new epitopes are not deleted nor inactivated by self-tolerance mechanisms. Our results present the first NY-BR-1-specific DRB1*0301- and HLA-DRB1*0401-restricted T cell epitopes that could be exploited for therapeutic intervention against breast cancer. (c) 2014 Wiley Periodicals, Inc.
    Type of Publication: Journal article published
    PubMed ID: 25387692
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  • 6
    Keywords: antigen presentation ; MHC MOLECULES ; innate immunity ; GENE-THERAPY ; RETROVIRAL VECTORS ; BROADLY NEUTRALIZING ANTIBODIES ; LEUKOCYTE ADHESION DEFICIENCY ; CROSS-SPECIES TRANSMISSION ; T-LYMPHOCYTE EPITOPE ; FELINE FOAMY
    Abstract: The use of whole viruses as antigen scaffolds is a recent development in vaccination that improves immunogenicity without the need for additional adjuvants. Previous studies highlighted the potential of foamy viruses (FVs) in prophylactic vaccination and gene therapy. Replication-competent FVs can trigger immune signaling and integrate into the host genome, resulting in persistent antigen expression and a robust immune response. Here, we explored feline foamy virus (FFV) proteins as scaffolds for therapeutic B and T cell epitope delivery in vitro. Infection- and cancer-related B and T cell epitopes were grafted into FFV Gag, Env, or Bet by residue replacement, either at sites of high local sequence homology between the epitope and the host protein or in regions known to tolerate sequence alterations. Modified proviruses were evaluated in vitro for protein steady state levels, particle release, and virus titer in permissive cells. Modification of Gag and Env was mostly detrimental to their function. As anticipated, modification of Bet had no impact on virion release and affected virus titers of only some recombinants. Further evaluation of Bet as an epitope carrier was performed using T cell epitopes from the model antigen chicken ovalbumin (OVA), human tyrosinase-related protein 2 (TRP-2), and oncoprotein E7 of human papillomavirus type 16 (HPV16E7). Transfection of murine cells with constructs encoding Bet-epitope chimeric proteins led to efficient MHC-I-restricted epitope presentation as confirmed by interferon-gamma enzyme-linked immunospot assays using epitope-specific cytotoxic T lymphocyte (CTL) lines. FFV infection-mediated transduction of cells with epitope-carrying Bet also induced T-cell responses, albeit with reduced efficacy, in a process independent from the presence of free peptides. We show that primate FV Bet is also a promising T cell epitope carrier for clinical translation. The data demonstrate the utility of replication-competent and -attenuated FVs as antigen carriers in immunotherapy.
    Type of Publication: Journal article published
    PubMed ID: 26397953
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  • 7
    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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  • 8
    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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  • 9
    Keywords: CANCER ; INHIBITION ; THERAPY ; DIFFERENTIATION ; ACCUMULATION ; MICE ; T-CELLS ; DOWN-REGULATION ; ASSOCIATION ; cytokines ; PROGRESSION ; inflammation ; DYSFUNCTION ; ARGININE METABOLISM ; CHAIN EXPRESSION
    Abstract: Tumor microenvironment is characterized by chronic inflammation represented by infiltrating leukocytes and soluble mediators, which lead to a local and systemic immunosuppression associated with cancer progression. Here, we used the ret transgenic spontaneous murine melanoma model that mimics human melanoma. Skin tumors and metastatic lymph nodes showed increased levels of inflammatory factors such as IL-1 beta, GM-CSF, and IFN-gamma, which correlated with tumor progression. Moreover, Gr1(+)CD11b(+) myeloid-derived suppressor cells (MDSCs), known to inhibit tumor reactive T cells, were enriched in melanoma lesions and lymphatic organs during tumor progression. MDSC infiltration was associated with a strong TCR zeta-chain down-regulation in all T cells. Coculturing normal splenocytes with tumor-derived MDSC induced a decreased T-cell proliferation and.-chain expression, verifying the MDSC immunosuppressive function and suggesting that the tumor inflammatory microenvironment supports MDSC recruitment and immunosuppressive activity. Indeed, upon manipulation of the melanoma microenvironment with the phosphodiesterase-5 inhibitor sildenafil, we observed reduced levels of numerous inflammatory mediators (e.g., IL-1 beta, IL-6, VEGF, S100A9) in association with decreased MDSC amounts and immunosuppressive function, indicating an anti-inflammatory effect of sildenafil. This led to a partial restoration of zeta-chain expression in T cells and to a significantly increased survival of tumor-bearing mice. CD8 T-cell depletion resulted in an abrogation of sildenafil beneficial outcome, suggesting the involvement of MDSC and CD8 T cells in the observed therapeutic effects. Our data imply that inhibition of chronic inflammation in the tumor microenvironment should be applied in conjunction with melanoma immunotherapies to increase their efficacy
    Type of Publication: Journal article published
    PubMed ID: 21969559
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  • 10
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