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  • 1
    Keywords: CELLS ; EXPRESSION ; Germany ; CLONING ; GENE ; PROTEIN ; PROTEINS ; transcription ; RELEASE ; DNA ; FAMILY ; DOMAIN ; CARCINOGENESIS ; CONTRAST ; PARTICLES ; virus ; DEGRADATION ; REPLICATION ; ANTIVIRAL ACTIVITY ; CONSTRUCTION ; IMMUNODEFICIENCY-VIRUS ; RE ; spumaretrovirus ; zoonosis ; cytidine deamination ; HIV-1 VIF ; HYPERMUTATION ; restriction factor ; virion infectivity factor
    Abstract: Genome hypermutation of different orthoretroviruses by cellular cytidine deaminases of the APOBEC3 family during reverse transcription has recently been observed. Lentiviruses like HIV-1 have acquired proteins preventing genome editing in the newly infected cell. Here we show that feline foamy virus (FFV), a typical member of the foamy retrovirus subfamily Spumaretrovirinae, is also refractory to genome deamination. APOBEC3-like FFV genome editing in APOBEC3-positive feline CRFK cells only occurs when the accessory FFV Bet protein is functionally inactivated. Editing of bet-deficient FFV genomes is paralleled by a strong decrease in FFV titer. In contrast to lentiviruses, cytidine deamination already takes place in APOBEC3-positive FFV-producing cells, because edited proviral DNA genomes are consistently present in released particles. By cloning the feline APOBEC3 orthologue, we found that its homology to the second domain of human APOBEC3F is 48%. Expression of feline APOBEC3 in APOBEC3-negative human 293T cells reproduced the effects seen in homologous CRFK cells: Bet-deficient FFV displayed severely reduced titers, high-level genome editing, reduced particle release, and suppressed Gag processing. Although WT Bet efficiently preserved FFV infectivity and genome integrity, it sustained particle release and Gag processing only when fe3 was moderately expressed. Similar to lentiviral Vif proteins, FFV Bet specifically bound feline APOBEC3. In particles from Bet-deficient FFV, feline APOBEC3 was clearly present, whereas its foamy viral antagonist Bet was undetectable in purified WT particles. This is the first report that, in addition to lentiviruses, the foamy viruses also developed APOBEC3-counteracting proteins
    Type of Publication: Journal article published
    PubMed ID: 15911774
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  • 2
    Keywords: RECEPTOR ; CANCER ; CELLS ; ENDOTHELIAL-CELLS ; EXPRESSION ; IN-VITRO ; BLOOD ; ENDOTHELIAL GROWTH-FACTOR ; Germany ; human ; INHIBITION ; KINASE ; TOXICITY ; PROTEIN ; DONOR ; ANTIGEN ; ASSAY ; PEPTIDES ; VACCINE ; EPITOPE ; IMMUNOTHERAPY ; T-LYMPHOCYTES ; GENE DELIVERY ; CYTOKINE ; ASSAYS ; cytotoxic T lymphocyte ; INHIBITS TUMOR ANGIOGENESIS
    Abstract: Antigen-specific cancer immunotherapy directed toward tumor-nourishing angiogenic blood vessels holds the promise of high efficacy, low toxicity, and ease of application. To evaluate whether the human angiogenic kinase insert domain-containing receptor (KDR) can serve as a target for cellular immunotherapy, 19 peptide sequences with HLA-A*0201 motifs were selected by computer-based algorithms. Five peptides (KDR82-90, KDR288-297, KDR766-774, KDR1093-1101, KDR1035-1044) stimulated specific cytotoxic T lymphocytes (CTLs) from peripheral-blood mono-nuclear cells (PBMCs) of 3 HLA-A*0201 donors. The decapeptide KDR288-297 was efficient in sensitizing target cells for recognition by a CTL clone at a concentration of 10 nM. More important, KDR288-297 specific CTLs lysed target cells transfected with HLA-A2/KDR cDNAs and a range of HLA-matched KDR+ angiogenic endothelial cells (aECs) and also recognized CD34(+) endothelial progenitor cells. The specificity of CTLs was further confirmed by tetramer assay and cold-target inhibition assay. In addition, ex vivo exposure of aECs to the inflammatory cyto-kines enhanced CTL reactivity, which is in keeping with up-regulated KDR and HLA class 1 expression. In Matrigel assays, recognition of aECs by specific CTLs triggered an antivascular effect. These findings provide the first proof of the antigenic property of KDR protein and may be useful for devising new immunotherapeutic approaches to human cancers. (Blood. 2006; 107:1476-1483) (c) 2006 by The American Society of Hematology
    Type of Publication: Journal article published
    PubMed ID: 16234362
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  • 3
    Keywords: CELLS ; CELL ; Germany ; INHIBITION ; PROTEIN ; PROTEINS ; COMPLEX ; MECHANISM ; DOMAIN ; FORM ; PARTICLES ; DEGRADATION ; ANTIVIRAL ACTIVITY ; HIV-1 VIF ; LEUKEMIA-VIRUS ; VIF ; 2 DISTINCT ; ANTIRETROVIRAL DEFENSE ; CYTIDINE DEAMINASES ; EDITING ENZYME APOBEC3G ; MURINE APOBEC3 ; SOCS-BOX ; TYPE-1 VIF
    Abstract: The APOBEC3 cytidine deaminases are part of the intrinsic defense of cells against retroviruses. Lentiviruses and spumaviruses have evolved essential accessory proteins, Vif and Bet, respectively, which counteract the APOBEC3 proteins. We show here that Bet of the Prototype foamy virus inhibits the antiviral APOBEC3C activity by a mechanism distinct to Vif: Bet forms a complex with APOBEC3C without inducing its degradation. Bet abolished APOBEC3C dimerization as shown by co-immunoprecipitation and cross-linking experiments. These findings implicate a physical interaction between Bet and the APOBEC3C. Subsequently, we identified the Bet interaction domain in human APOBEC3C in the predicted APOBEC3C dimerization site. Taken together, these data support the hypothesis that Bet inhibits incorporation of APOBEC3Cs into retroviral particles. Bet likely achieves this by trapping APOBEC3C protein in complexes rendering them unavailable for newly generated viruses due to direct immobilization
    Type of Publication: Journal article published
    PubMed ID: 19074429
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  • 4
    Abstract: We present a flexible and highly specific targeting method for lentiviral vectors based on single-chain antibodies recognizing cell-surface antigens. We generated lentiviral vectors specific for human CD105(+) endothelial cells, human CD133(+) hematopoietic progenitors and mouse GluA-expressing neurons. Lentiviral vectors specific for CD105 or for CD20 transduced their target cells as efficiently as VSV-G pseudotyped vectors but discriminated between endothelial cells and lymphocytes in mixed cultures. CD133-targeted vectors transduced CD133(+) cultured hematopoietic progenitor cells more efficiently than VSV-G pseudotyped vectors, resulting in stable long-term transduction. Lentiviral vectors targeted to the glutamate receptor subunits GluA2 and GluA4 exhibited more than 94% specificity for neurons in cerebellar cultures and when injected into the adult mouse brain. We observed neuron-specific gene modification upon transfer of the Cre recombinase gene into the hippocampus of reporter mice. This approach allowed targeted gene transfer to many cell types of interest with an unprecedented degree of specificity.
    Type of Publication: Journal article published
    PubMed ID: 20935652
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  • 5
    Abstract: BACKGROUND: The non-pathogenic course of SIV infection in its natural host is characterized by robust viral replication in the absence of chronic immune activation and T cell proliferation. In contrast, acutely lethal enteropathic SIVsmm strain PBj induces a strong immune activation and causes a severe acute and lethal disease in pig-tailed macaques after cross-species transmission. One important pathogenicity factor of the PBj virus is the PBj-Nef protein, which contains a conserved diacidic motif and, unusually, an immunoreceptor tyrosine-based activation motif (ITAM). RESULTS: Mutation of the diacidic motif in the Nef protein of the SIVsmmPBj abolishes the acute phenotype of this virus. In vitro, wild-type and mutant PBj (PBj-Nef202/203GG) viruses replicated to similar levels in macaque PBMCs, but PBj-Nef202/203GG no longer triggers ERK mitogen-activated protein (MAP) kinase pathway including an alteration of a Nef-associated Raf-1/ERK-2 multiprotein signaling complex. Moreover, stimulation of IL-2 and down-modulation of CD4 and CD28 were impaired in the mutant virus. Pig-tailed macaques infected with PBj-Nef202/203GG did not show enteropathic complications and lethality as observed with wild-type PBj virus, despite efficient replication of both viruses in vivo. Furthermore, PBj-Nef202/203GG infected animals revealed reduced T-cell activation in periphery lymphoid organs and no detectable induction of IL-2 and IL-6. CONCLUSIONS: In sum, we report here that mutation of the diacidic motif in the PBj-Nef protein abolishes disease progression in pig-tailed macaques despite efficient replication. These data suggest that alterations in the ability of a lentivirus to promote T cell activation and proliferation can have a dramatic impact on its pathogenic potential.
    Type of Publication: Journal article published
    PubMed ID: 21366921
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  • 6
    Abstract: To target oncolytic measles viruses (MV) to tumors, we exploit the binding specificity of designed ankyrin repeat proteins (DARPins). These DARPin-MVs have high tumor selectivity while maintaining excellent oncolytic potency. Stability, small size, and efficacy of DARPins allowed the generation of MVs simultaneously targeted to tumor marker HER2/neu and cancer stem cell (CSC) marker EpCAM. For optimization, the linker connecting both DARPins was varied in flexibility and length. Flexibility had no impact on fusion helper activity whereas length had. MVs with bispecific MV-H are genetically stable and revealed the desired double-target specificity. In vitro, the cytolytic activity of bispecific MVs was superior or comparable to mono-targeted viruses depending on the target cells. In vivo, therapeutic efficacy of the bispecific viruses was validated in an orthotopic ovarian carcinoma model revealing an effective reduction of tumor mass. Finally, the power of bispecific targeting was demonstrated on cocultures of different tumor cells thereby mimicking tumor heterogeneity in vitro, more closely reflecting real tumors. Here, bispecific excelled monospecific viruses in efficacy. DARPin-based targeting domains thus allow the generation of efficacious oncolytic viruses with double specificity, with the potential to handle intratumoral variation of antigen expression and to simultaneously target CSCs and the bulk tumor mass.
    Type of Publication: Journal article published
    PubMed ID: 27119117
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  • 7
    Keywords: RECEPTOR ; CELLS ; EXPRESSION ; INHIBITOR ; CELL ; Germany ; human ; MODEL ; MODELS ; THERAPY ; SUPPORT ; PROTEIN ; PROTEINS ; cell line ; LINES ; RELEASE ; INFECTION ; CONTRAST ; T cell ; T-CELL ; CELL-LINES ; TYPE-1 ; SUSCEPTIBILITY ; ENTRY ; PARTICLES ; virus ; TRANSGENIC MICE ; VECTORS ; VECTOR ; CELL-LINE ; LINE ; EFFICIENT ; PATHOGENESIS ; US ; POLYPEPTIDE ; MURINE CELLS ; REPLICATION ; vaccination ; INFECTIVITY ; cell lines ; LUCIFERASE ; HUMAN-IMMUNODEFICIENCY-VIRUS ; CD4(+) T-CELLS ; ORIGIN ; INHIBITORS ; ANIMAL-MODELS ; THERAPIES ; VIRUS-INFECTION ; HIV ; ANIMAL-MODEL ; USA ; EVALUATE ; IMMUNODEFICIENCY VIRUS ; INHIBIT ; animal ; microbiology ; animal model ; HUMAN MONOCYTES ; viral ; virology ; block ; CD4 RECEPTOR ; SOLUBLE CD4 ; OCCURS ; AIDS-LIKE DISEASE ; animal models ; ENZYME APOBEC3G ; HIV-1 INFECTION ; SCID-HU MOUSE ; VIF PROTEIN
    Abstract: The productive replication of human immunodeficiency virus type 1 (HIV-1) occurs exclusively in defined cells of human or chimpanzee origin, explaining why heterologous animal models for HIV replication, pathogenesis, vaccination, and therapy are not available. This lack of an animal model for HIV-1 studies prompted us to examine the susceptibility of feline cells in order to evaluate the cat (Felis catus) as an animal model for studying HIV-1. Here, we report that feline cell lines harbor multiple restrictions with respect to HIV-1 replication. The feline CD4 receptor does not permit virus infection. Feline T-cell lines MYA-1 and FeT-1C showed postentry restrictions resulting in low HIV-1 luciferase reporter activity and low expression of viral Gag-Pol proteins when pseudotyped vectors were used. Feline fibroblastic CrFK and KE-R cells, expressing human CD4 and CCR5, were very permissive for viral entry and HIV-long terminal repeat-driven expression but failed to support spreading infection. KE-R cells displayed a profound block with respect to release of HIV-1 particles. In contrast, CrFK cells allowed very efficient particle production; however, the CrFK cell-derived HIV-1 particles had low specific infectivity. We subsequently identified feline apolipoprotein B-editing catalytic polypeptide 3 (feAPOBEC3) proteins as active inhibitors of HIV-1 particle infectivity. CrFK cells express at least three different APOBEC3s: APOBEC3C, APOBEC3H, and APOBEC3CH. While the feAPOBEC3C did not significantly inhibit HIV-1, the feAPOBEC3H and feAPOBEC3CH induced G to A hypermutations of the viral cDNA and reduced the infectivity similar to 10- to similar to 40-fold
    Type of Publication: Journal article published
    PubMed ID: 17459941
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  • 8
    Keywords: INHIBITOR ; Germany ; human ; SUPPORT ; HISTORY ; SITE ; SITES ; GENE ; GENES ; GENOME ; PROTEIN ; PROTEINS ; COMPLEX ; COMPLEXES ; SEQUENCE ; SEQUENCES ; virus ; genetics ; leukemia ; HUMAN GENOME ; EVOLUTION ; REPLICATION ; SELECTION ; foamy virus ; heredity ; IMMUNODEFICIENCY-VIRUS TYPE-1 ; INHIBITORS ; MULTIPLE SEQUENCE ALIGNMENT ; ASSEMBLIES ; assembly ; PHYLOGENETIC ANALYSIS ; rodents ; virion infectivity factor ; TRANSMISSION ; LEUKEMIA-VIRUS ; microbiology ; ENGLAND ; EXPANSION ; host ; biotechnology ; ENZYME APOBEC3G ; mammals ; ADAPTIVE EVOLUTION ; MAXIMUM-LIKELIHOOD
    Abstract: Background: Over the past years a variety of host restriction genes have been identified in human and mammals that modulate retrovirus infectivity, replication, assembly, and/or cross-species transmission. Among these host-encoded restriction factors, the APOBEC3 (A3; apolipoprotein B mRNA-editing catalytic polypeptide 3) proteins are potent inhibitors of retroviruses and retrotransposons. While primates encode seven of these genes (A3A to A3H), rodents carry only a single A3 gene. Results: Here we identified and characterized several A3 genes in the genome of domestic cat (Felis catus) by analyzing the genomic A3 locus. The cat genome presents one A3H gene and three very similar A3C genes (a-c), probably generated after two consecutive gene duplications. In addition to these four one-domain A3 proteins, a fifth A3, designated A3CH, is expressed by readthrough alternative splicing. Specific feline A3 proteins selectively inactivated only defined genera of feline retroviruses: Bet-deficient feline foamy virus was mainly inactivated by feA3Ca, feA3Cb, and feA3Cc, while feA3H and feA3CH were only weakly active. The infectivity of Vif-deficient feline immunodeficiency virus and feline leukemia virus was reduced only by feA3H and feA3CH, but not by any of the feA3Cs. Within Felidae, A3C sequences show significant adaptive selection, but unexpectedly, the A3H sequences present more sites that are under purifying selection. Conclusion: Our data support a complex evolutionary history of expansion, divergence, selection and individual extinction of antiviral A3 genes that parallels the early evolution of Placentalia, becoming more intricate in taxa in which the arms race between host and retroviruses is harsher
    Type of Publication: Journal article published
    PubMed ID: 18315870
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  • 9
    Keywords: CANCER ; CELLS ; ENDOTHELIAL-CELLS ; GROWTH ; GROWTH-FACTOR ; tumor ; TUMOR-CELLS ; CELL ; Germany ; human ; MODEL ; MODELS ; ALGORITHM ; PROTEIN ; PATIENT ; RESPONSES ; MECHANISM ; ANTIGEN ; DENDRITIC CELLS ; mechanisms ; T cell ; T cells ; T-CELL ; T-CELLS ; BINDING ; ASSOCIATION ; MOLECULE ; culture ; RECOGNITION ; STIMULATION ; DESIGN ; LYMPHOCYTES ; REGION ; VACCINES ; VACCINE ; STRATEGIES ; CD8(+) ; IMMUNITY ; IMMUNOTHERAPY ; T-LYMPHOCYTES ; T lymphocyte ; experimental design ; HEALTHY ; ANTITUMOR IMMUNITY ; T lymphocytes ; ANIMAL-MODELS ; ONCOLOGY ; RE ; TUMOR-GROWTH ; monitoring ; DNA vaccine ; FACTOR RECEPTOR-1 ; ALLELES ; dendritic cell ; ANIMAL-MODEL ; USA ; vascular endothelial growth factor ; CANDIDATE ; cancer research ; CANCER-TREATMENT ; CANCERS ; animal ; animal model ; SHORT-TERM ; ENDOTHELIAL GROWTH ; animal models ; CELL RESPONSES ; DONORS ; IMMUNOGENE THERAPY ; ACTIVE IMMUNOTHERAPY ; ANGIOGENESIS INHIBITORS ; MODEL ANTIGEN
    Abstract: Purpose: Given the multiple escape mechanisms of tumor cells, immunotherapy targeting tumor-dependent stroma may be an effective cancer treatment strategy. Animal models indicate that inducing immunity to tumor enclothelia engenders potent antitumor effects without significant pathology. Recently, the first human tumor enclothelial antigen vascular enclothelial growth factor receptor-2 (VEGFR-2) recognized by HLA class I-restricted CD8(+) T cells has been characterized. In this study, we sought to investigate specific recognition of this molecule by human CDC+ Tcells. Experimental Design: To identify HLA-DR-restricted antigenic pepticles on VEGFR-2 recognized by CDC Tcells of healthy donors and cancer patients. Results: Nine candidate VEGFR-2 pepticles with high binding probability to six common HLA-DRB1 alleles were synthesized using the SYFPEITHI algorithm. One 15-mer pepticle (EKRFVPDGNRISWDS), mapping to the 167-181 region of VEGFR-2, stimulated CDC Tcells in association with several HLA-DR alleles, including DR4 and DR7. Importantly, the epitope could be naturally processed and presented both by HILA-DR-matched antigen-expressing proliferating endothelial cells and by dendritic cells loaded with the native antigen. Furthermore, circulating VEGFR-2- specific CDC T cells were detected in 4 of 10 healthy donors and 12 of 40 cancer patients even after single-round pepticle stimulation in short-term culture. Patient's T cells could recognize a ntigen-expressing proliferating enclothelial cells in a HLA-DR-restricted fashion. Conclusion: These findings indicate an important role for the 167-181 region of VEGFR-2 in the stimulation of CD4(+) T cell responses to VEGFR-2 protein, and may be instrumental both for the development and monitoring of upcoming antitumor vessel vaccines against different cancers based on VEGFR-2 immunogens
    Type of Publication: Journal article published
    PubMed ID: 18594014
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  • 10
    Keywords: gene therapy ; AAV ; lentiviral ; GMP
    Abstract: In the past 5 years, European investigators have played a major role in the development of clinical gene therapy. The provision of substantial funds by some individual member states to construct GMP facilities makes it an opportune time to network available gene therapy GMP facilities at an EU level. The integrated coordination of GMP production facilities and human skills for advanced gene and genetically-modified (GM) cell therapy, can dramatically enhance academic-led "First-in-man" gene therapy trials. Once proof of efficacy is gathered, technology can be transferred to the private sector which will take over further development taking advantage of knowledge and know-how. Complex technical challenges require existing production facilities to adapt to emerging technologies in a coordinated manner. These include a mandatory requirement for the highest quality of production translating gene-transfer technologies with pharmaceutical-grade GMP processes to the clinic. A consensus has emerged on the directions and priorities to adopt, applying to advanced technologies with improved efficacy and safety profiles, in particular AAV, lentivirus-based and oncolytic vectors. Translating cutting-edge research into "First-in-man" trials require that pre-normative research is conducted which aims to develop standard assays, processes and candidate reference materials. This research will help harmonise practices and quality in the production of GMP vector lots and GM-cells. In gathering critical expertise in Europe and establish conditions for interoperability, the PEVI infrastructure will contribute to the demands of the advanced therapy medicinal products* regulation and to both health and quality of life of EU-citizens.
    Type of Publication: Journal article published
    PubMed ID: 21054249
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