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  • 1
    Keywords: CELLS ; CELL ; Germany ; GENE ; PROTEIN ; PROTEINS ; EFFICIENCY ; ANTIGEN ; culture ; PARTICLES ; virus ; VECTORS ; PRODUCT ; PURIFICATION ; MAMMALIAN-CELLS ; VACCINE ; STRATEGIES ; NETHERLANDS ; vaccination ; CALCIUM ; GENE DELIVERY ; GENE-PRODUCT ; SCALE ; TRANSFECTION ; cost ; HIV ; methods ; ENV ; microbiology ; virology ; TRANSIENT TRANSFECTION ; biotechnology ; HIV pseudovirions ; HIV vaccine ; pseudovirion ; DNA DELIVERY ; DNA transfection ; polyethylenimine ; pseudovirions
    Abstract: HIV vaccine strategies which employ pseudovirions (PVs) as the source of antigen require large amounts of particles. These are typically generated by transient transfection of mammalian cells and purification of the released PVs from the culture supernatant. Since efficiency and cost of transfection are key issues, in this report the transfection efficiencies, achieved by employing a panel of high-molecular-weight linear polyethylenimines (PEIs) and small cross-linked PEIs, were analyzed and compared to those obtained by transfections with calcium phosphate or the commercial reagent Polyfect (R). High efficiencies were obtained using several of the modified PEIs, and the transfections with these inexpensive reagents were very robust. The observed efficiencies (as quantitated by amounts of expressed gene product) were two to four fold superior to calcium phosphate transfection and approximately equal to that achieved using Polyfect (R) which is, however, prohibitively expensive for large scale applications. An optimized and rapid protocol for the large scale production and purification of HIV-PVs from 293T cells growing in so-called cell stacks and transfected with the best reagent identified, PEI87, is described here. The generated PVs, obtained with a yield in the range of 0.4 mg virion-associated HIV-CA/liter culture supernatant, exhibited only very minimal contamination with non-viral proteins and were thus suitable for vaccination applications. (c) 2007 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17719656
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  • 2
    Keywords: Germany ; human ; EPIDEMIOLOGY ; POPULATION ; SAMPLE ; SAMPLES ; DNA ; SKIN ; virus ; PCR ; human papillomavirus ; HPV ; BETA ; NETHERLANDS ; PREVALENCE ; papillomaviruses ; EPIDERMODYSPLASIA-VERRUCIFORMIS ; hair ; ACTINIC KERATOSES ; HUMAN PAPILLOMAVIRUSES ; virology ; NOV ; biotechnology ; HAIRS ; Detection ; Beta papillornavirus ; Cellular DNA ; EYEBROW HAIRS ; Multiplicity
    Abstract: In view of the low loads of beta human papillomaviruses in skin samples, amounts of cellular DNA used in qualitative PCR may become limiting for virus detection and introduce variations in prevalence and multiplicity. This issue was explored within the context of a multicentre study and increasing prevalence and multiplicity was found with increasing input amounts of cellular DNA extracted from hair bulbs. To improve the quality and comparability between different epidemiologic studies ideally equal amounts of cellular DNA should be employed. When cellular DNA input varies this should be clearly taken into account in assessing viral prevalence and multiplicity. (C) 2009 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19591874
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  • 3
    Keywords: CELLS ; EXPRESSION ; DNA ; SKIN ; BOVINE PAPILLOMAVIRUS ; genotyping ; PREVALENCE ; ALIGNMENT ; SARCOIDS ; CATTLE ; GENOMES ; POSITION ; BOVINE PAPILLOMAVIRUS TYPE-1 ; BPV ; BSBP-PCR/MBG ; Multiple infection
    Abstract: Bovine papillomaviruses (BPV) induce benign tumours of the cutaneous or mucosal epithelia in cattle, but are also involved in the development of cancer of the urinary bladder and of the upper gastrointestinal tract. Current BPV genotyping assays employ techniques developed originally for the detection of human papillomaviruses. These methods rely on consensus PCR amplification and subsequent sequencing and are cumbersome and limited in their analytic sensitivity to detect BPV, especially in multiple infections. In this study, a novel multiplex BPV genotyping assay is described to detect sensitively and specifically BPV-1 to -10 as well as BaPV-11. The assay is based on a multiplex PCR using novel broad-spectrum bovine papillomavirus (BSBP) primers followed by multiplex bovine genotyping (MBG) by Luminex xMAP technology. The detection limit of the assay was shown to be between 10 and 100 BPV genomes. In a first application, BPV was detected in 100% of wart preparations with BPV-8 being most prevalent, followed by types 6, 1 and 10. The majority of warts were positive for at least four BPV types. In conclusion, BSBP-PCR/MBG is a powerful high-throughput method suitable for the study of the natural history of BPV and could be useful to veterinarians for the monitoring of the efficacy of future BPV vaccines.
    Type of Publication: Journal article published
    PubMed ID: 20816698
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  • 4
    Keywords: DNA ; PCR ; SQUAMOUS-CELL CARCINOMA ; SKIN-CANCER ; EPIDERMODYSPLASIA-VERRUCIFORMIS ; E6 PROTEINS ; organ transplant recipients ; LINE-BLOTTING SYSTEM ; GENOMIC CHARACTERIZATION ; BETAPAPILLOMAVIRUS INFECTION
    Abstract: Cutaneous human papillomavirus (HPV) may play a role in the development of cutaneous squamous cell carcinoma. HPV copy numbers in cutaneous squamous cell carcinoma are very low and hence sensitive and reliable detection methods are important, particularly to examine the natural history of cutaneous HPV. In the present study, the presence of cutaneous HPV types was examined in 194 skin swabs and in a subgroup of 91 skin swabs, and compared using three different PCR based methods: (i) beta/gamma cutaneous HPV PCR reverse-line-blotting (BGC-PCR RLB), (ii) multiplex cutaneous papillomavirus genotyping (McPG) and (iii) FAP PCR. The HPV prevalence was 75% (68/91) with BGC-PCR RLB, 64% (124/194) with McPG and 72% (139/194) with FAP PCR. The agreement for the detection of HPV between the three methods in the subset of 91 samples was 73% (66/91; kappa=0.34) for BGC-PCR RLB and McPG, 75% (68/91; kappa=0.32) for BGC-PCR RLB and FAP PCR, and 69% (63/91; kappa=0.25) for McPG and FAP PCR. For McPG and FAP PCR, 194 specimens were tested in total, with an overall agreement of 66% (129/194; kappa=0.24) for the detection of HPV. The concordance between the three methods was moderate, which could be explained by different HPV types detectable with each method; the high number of multiple infections and the low viral copy number in human skin. Overall, many cutaneous HPV types were identified and multiple HPV types were found frequently in the human skin swabs.
    Type of Publication: Journal article published
    PubMed ID: 23124002
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  • 5
    Keywords: LESIONS ; ASSAY ; WOMEN ; CERVICAL-CANCER ; HIGH-RISK ; POLYMERASE-CHAIN-REACTION ; NATURAL-HISTORY ; VIRAL LOAD ; MULTIPLEX PCR ; HUMAN-PAPILLOMAVIRUS TYPE-6
    Abstract: HPV 16 and HPV 18 are responsible for more than 75% of cervical cancers and high HPV 16 loads are associated with both prevalent and incident lesions. The objective of the present study was to develop a method allowing the detection and quantitation of HPV 16 and 18 DNA to improve future strategies for cervical cancer screening. A duplex real-time PCR allowing the simultaneous quantitation of both HPV 16 and HPV 18 was carried out. Mixes of HPV 16 and HPV 18 whole genome plasmids were prepared to test a wide range of viral DNA concentrations. The values obtained for each mix of plasmids with the simplex and the duplex PCR were very close to the theoretical values except when a HPV type represented only 1:1000 genome equivalent or lower than the concurrent type. Cervical samples harboring HPV 16, HPV 18 or both types were tested by comparing the results with simplex and duplex real-time PCR assays. HPV 16 and HPV 18 genome titers were similar with the two assays. In conclusion, the real-time duplex PCR proved to be robust for HPV 16 and HPV 18 DNA quantitation.
    Type of Publication: Journal article published
    PubMed ID: 23891872
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  • 6
    Keywords: PROTEIN ; SEQUENCE ; ASSOCIATION ; TYPE-16 ; bioinformatics ; PREDICTION ; HPV-16 E6 ; INDIAN WOMEN ; NATURAL VARIANTS ; HPV16 E6
    Abstract: The infection with high-risk human papillomavirus (HR-HPV) is the most important risk factor for development of cervical cancer. The intra-type variations of HPV have different biological and pathological consequences with respect to disease progression. In the present study, six major Indian variants were experimentally identified in E6 gene of HPV-16 and showed their impact on immunogenicity by in silico methods. Four different phylogenetic lineages were observed in sequences including European (E) prototype, European variant, Asian and American Asian variant classes and complete absence of African phylogenetic lineages. On the prediction of B- and T-cell epitopes, 18 and 23 potent epitopes for MHC-II alleles, 10 potent MHC-I and 15 B-cell epitopes in each reference and variant sequence were identified. Interestingly, the presence of variation H78Y and L83V result in creation of four new epitopes for the HLA-DQA1*0101/DQB1*0501. Out of 15 B-cell predicted epitopes, three most potent epitopes were identified in both reference and variant sequence. Notably the amino acid stretch from amino acid 16-60 and 76-94 are very important for the immunological properties of E6 protein because these regions contain majority of the predicted epitopes. In future, this could control the cervical cancer by targeting these amino acid stretches for the development of HPV-16 vaccine.
    Type of Publication: Journal article published
    PubMed ID: 25800725
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  • 7
  • 8
    Keywords: CELLS ; tumor ; CELL ; Germany ; THERAPY ; TOOL ; CLONING ; DISTINCT ; GENE ; PROTEIN ; PROTEINS ; MONOCLONAL-ANTIBODY ; gene therapy ; gene transfer ; GENE-TRANSFER ; antibodies ; antibody ; PARTICLES ; FOCI ; IDENTIFICATION ; VECTORS ; VECTOR ; CAPSID PROTEIN ; MONOCLONAL-ANTIBODIES ; NETHERLANDS ; PREVALENCE ; GENE-THERAPY ; AAV ; ELISA ; RECOMBINANT ; PROGRAM ; RE ; ADENOASSOCIATED VIRUS TYPE-2 ; SERUM ANTIBODIES ; development ; methods ; monoclonal antibodies ; capsid ; TOOLS ; microbiology ; CAPTURE ELISA ; virology ; serotype ; Capsid Proteins
    Abstract: Adeno-associated viruses (AAV) have been developed and evaluated as recombinant vectors for gene therapy. More recently, due to the advantages they offer for gene transfer, several AAV serotypes have gained increasing interest. However, monoclonal antibodies for the characterization and quantitation of vectors derived from different serotypes are at present not available. Serotype-specific monoclonal antibodies (mAbs) against the capsids of the serotypes 1/6, 4 and 5 are describe1 a and b cross-reacted with its highly related AAV6 serotype, but not with the other serotypes tested. The new antibodies recognized exclusively assembled capsids and neither free nor denatured capsid proteins as shown by fractionation experiments. In immunofluorescence experiments, the mAbs stained only distinct intranuclear foci in cells expressing the capsid protein. The development of capture E-LISAs for quantitation of AAV1 and 6, AAV4 or AAV5 capsids illustrates that these novel monoclonal antibodies provide valuable tools for characterization of vector stocks. (c) 2006 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 17126418
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  • 9
    Keywords: CANCER ; tumor ; Germany ; MODEL ; COHORT ; PROTEIN ; PROTEINS ; TUMORS ; INFECTION ; CARCINOGENESIS ; ANTIGEN ; SKIN ; papillomavirus ; SEQUENCE ; antibodies ; antibody ; REPRODUCIBILITY ; skin cancer ; NETHERLANDS ; IMMUNE-RESPONSE ; L1 ; INFECTIONS ; PREVALENCE ; rodent ; glutathione-S-transferase ; papillomaviruses ; SCIENCE ; development ; SQUAMOUS-CELL ; serology ; NATALENSIS PAPILLOMAVIRUS ; HEALTHY SKIN ; viruses ; MnPV ; IMMUNE ; CELL CARCINOMAS ; GST-capture ELISA ; Mastomys ; McPV2
    Abstract: The present report describes the development of an enzyme-linked immunosorbent assay (ELISA), whereby the first insights have been obtained into the humoral immune response to papillomavirus (PV) infections of the rodent Mastomys coucha, a natural model for papillomavirus-induced skin carcinogenesis. The established glutathione S-transferase (GST)-capture ELISA is based on a one-step purification of bacterially expressed antigens and was designed to detect serum antibodies against L1 capsid proteins of Mastomys natalensis papillomavirus (MnPV) and M. coucha papillomavirus 2 (McPV2). Both viruses are spread widely within the colony at the German Cancer Research Center. The animals are unique in that they spontaneously develop multiple epithelial tumors such as MnPV-induced papillomas and McPV2-induced condylomas. The humoral immune response of a cohort of 98 Mastomys was analysed and revealed a high prevalence of antibodies to L1 (MnPV: 36.7%, McPV2: 52.0%). Furthermore, the seroreactivity to both viruses was significantly increased in animals with the respective tumors (22 papillomas, 21 condylomas) as compared to 55 tumor-free controls (MnPV: p 〈 0.0001; McPV2: p = 0.0022). The identified assay conditions showed a high level of sensitivity (MnPV: 75.7%; McPV2: 85.7%) and reproducibility (MnPV: R-2 = 0.83; McPV2: R-2 = 0.79), validating the GST-capture ELISA as a powerful method for monitoring papillomavirus serology in M. coucha. (C( 2009 Elsevier B.V. All rights reserved
    Type of Publication: Journal article published
    PubMed ID: 19800367
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  • 10
    Keywords: CELLS ; EXPRESSION ; GENE ; PROTEIN ; PROTEINS ; LIGAND ; INFECTION ; ANTIGEN ; SEQUENCE ; antibodies ; antibody ; MOUSE ; IDENTIFICATION ; PRODUCT ; MONOCLONAL-ANTIBODIES ; TRAIL ; immunoassay ; ABSENCE ; PRODUCTS ; INSECT CELLS ; ENVELOPE GLYCOPROTEIN ; CULTURES ; NUCLEAR POLYHEDROSIS-VIRUS ; V-CATH ; mAb ; baculovirus AcMNPV ; Sf9 insect cells ; mAb CD95(APO-1/Fas) ligand
    Abstract: 0720,english,Production of different recombinant proteins in baculovirus AcMNPV (BV)-infected cells may be facilitated by the availability of immunoassays to monitor active infection of Sf9 insect cells. To this end, two hybridomas secreting mouse monoclonal antibodies (mAbs) were established to different BV-related products. The proteins recognized by mAb SM22 and SM62 were easily detectable by immunoblotting and immunostaining in Sf9 cells infected with recombinant BV (rBV), but not in non-infected cells. Their production paralleled that of the recombinant proteins analyzed but was independent of the type of recombinant protein expressed. Thus, immunoassays with these mAbs allow: (1) daily monitoring of the infection occurring in small and large scale cultures of Sf9 cells using a defined rBV; (2) preliminary assessment of active rBV infection in the absence of a specific reagent for the recombinant protein and (3) single-reagent comparison of the infection achieved in Sf9 cells exposed to rBVs expressing differen t recombinant proteins.
    Type of Publication: Journal article published
    PubMed ID: 9504747
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