T Hämmerle

IST Austria, Klosterneuberg, Lower Austria, Austria

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Publications (14)40.93 Total impact

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    ABSTRACT: A new 10% liquid human intravenous immunoglobulin (US trade name: Gammagard Liquid; European trade name: KIOVIG) manufactured by a process with three dedicated pathogen inactivation/removal steps (solvent/detergent treatment, 35-nm nanofiltration and low pH/elevated temperature incubation) was developed. The ability of the manufacturing process to inactivate/remove viruses and prions was investigated. Virus and prion removal capacities were assessed with down-scale spiking experiments, validated for equivalence to the large-scale process. Lipid-enveloped viruses were completely inactivated/removed by each of the three dedicated virus clearance steps, and for human immunodeficiency virus 1 (HIV-1) and pseudorabies virus (PRV), also by the upstream cold ethanol fractionation step. Relevant non-enveloped viruses [i.e. hepatitis A virus (HAV) and parvovirus B19 (B19V)] were effectively removed by nanofiltration and the cold ethanol fractionation step, and partial inactivation of non-enveloped viruses was achieved by low pH incubation. Overall log reduction factors were > 20.0 for HIV-1, > 18.1 for bovine viral diarrhoea virus, > 16.3 for West Nile virus, > 10.0 for influenza A virus subtype H5N1, > 21.8 for PRV, 12.0 for HAV, > 12.1 for encephalomyocarditis virus, 10.6 for B19V and 10.3 for mice minute virus. Prions (Western blot assay) were completely removed (> or = 3.2 mean log reduction) by a step of the cold ethanol fractionation process. Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.
    Vox Sanguinis 04/2008; 94(3):184-92. DOI:10.1111/j.1423-0410.2007.01016.x · 3.30 Impact Factor
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    ABSTRACT: Filters with nominal pore sizes in the nanometer range are well-established tools for enhancing the virus safety margins of plasma-derived products, yet intrinsically less successful for smaller viruses such as hepatitis A virus (HAV) and human parvovirus B19 (B19V). The formation of virus-antibody complexes increases the effective size of these smaller viruses and would thus improve their removal by nanofiltration. While the principle of virus removal by antibody-dependent nanofiltration has been demonstrated with animal antisera and viruses spiked into human plasma product intermediates, the significance of these results remains unclear due to the potential contributions of xenoanti-bodies and/or heteroagglutination in such heterologous systems. The current study investigated antibody-dependent virus removal by nanofiltration in a heterologous animal parvovirus system to establish the concentration dependence of the effect. In addition, the phenomenon was investigated in a homologous system with custom-made HAV and B19V antibody-free and -containing human immunoglobulin intermediates. Viruses were analyzed with infectivity assays and fully validated polymerase chain reaction assays that also circumvent the obscuring effects of neutralizing antibodies with infectivity assays. By use of the heterologous mice minute virus and the homologous HAV and B19V systems, viruses passed the 35-nm (Planova 35N) filter in the absence of specific antibodies. Beyond a threshold virus antibody concentration, nanofiltration resulted in effective virus removal of viruses smaller than the nominal pore size of the filter used. HAV and B19V are effectively removed by antibody-dependent 35N nanofiltration, already at intermediate antibody concentrations well below those comparable to human plasma pools for fractionation.
    Transfusion 08/2006; 46(7):1143-51. DOI:10.1111/j.1537-2995.2006.00864.x · 3.57 Impact Factor
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    ABSTRACT: Evidence is presented demonstrating that the distribution of data obtained applying a given RT-PCR method deviates from a normal distribution depending on the limit of detection. The effect of this is a bias towards higher values and concomitantly a systematic error in respect to the accuracy of the evaluation due to this deviation from normality. In addition, evidence is presented that an evaluation assuming a log-normal distribution is more appropriate.
    Molecular and Cellular Probes 09/2003; 17(4):171-4. DOI:10.1016/S0890-8508(03)00049-5 · 1.86 Impact Factor
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    ABSTRACT: Recombinant vaccinia viruses that express defective retroviral vectors upon a single infection event in normal host cells were constructed. The gag-pol and envelope genes and a retroviral vector unit were inserted as vaccinia virus promoter-controlled transcription units at three separate loci. The triple recombinant virus was used to infect such diverse cell types as monkey and rabbit kidney, human lung, and primary chicken cells, resulting in the production of transduction-competent defective retroviral vectors. Infection of Chinese hamster ovary cells, which are nonpermissive for vaccinia virus replication, also resulted in production of retroviral vectors and secondary permanent transduction of the host cells. Since vaccinia virus supports the expression of cytotoxic proteins, the vesicular stomatitis virus G glycoprotein could be chosen as the envelope allowing a broad host range of transduction. Functionality of particles was monitored by expression of the green fluorescent protein in transduced 3T3 cell clones. This is the first description of a single chimeric virus encoding and releasing functional retroviral vectors, providing proof of principle of the new concept. No replication-competent retrovirus was detectable by sensitive reverse transcriptase assays. Since vaccinia virus has a broad host range, is extremely robust, and can be obtained at high titers and safe nonreplicating vaccinia virus strains are available, the hybrid system may open new perspectives for gene delivery.
    Journal of Virology 07/2003; 77(12):7017-25. DOI:10.1128/JVI.77.12.7017-7025.2003 · 4.65 Impact Factor
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    F Gruber, F G Falkner, F Dorner, T Hämmerle
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    ABSTRACT: A real-time PCR method was developed to quantitate viral DNA that includes duplex amplification, internal standardization, and two-color fluorescence detection without the need to generate an external standardization curve. Applied to human parvovirus B19 DNA, the linear range was from 10(2) to at least 5 x 10(6) copies per ml of sample. The coefficient of variation was 0.29 using a run control of 2,876 copies per ml. The method reduces the risk of false-negative results, yields high precision, and is applicable for other DNA targets.
    Applied and Environmental Microbiology 07/2001; 67(6):2837-9. DOI:10.1128/AEM.67.6.2837-2839.2001 · 3.95 Impact Factor
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    ABSTRACT: A quantitative and systematic analysis is provided for ubiquitously present template DNA interfering with the quantification of human DNA by PCR. Two sources contributing to DNA background were identified. The first one is interpreted as DNA present in chemicals and on equipment and the second as caused by operator handling. The amounts were equivalent to 2.5 and 8.9 pg per mL of sample, and the estimated frequencies of contamination were 65 and 35%, respectively, resulting in an effective limit of detection of 17.4 pg/mL. Below this level--named effective laboratory background--a result could not be considered as authentic. Knowledge of these parameters is important for laboratories that analyze minute amounts of human DNA by PCR for purposes such as quantification, typing, and sequencing.
    Journal of Forensic Sciences 12/2000; 45(6):1307-11. · 1.31 Impact Factor
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    ABSTRACT: The primary objective of this study was to expand the safety and immunogenicity database of recombinant gp160 as a therapeutic vaccine in the treatment of HIV-infection. Preliminary efficacy data was also sought. This trial was a randomized, double-blind, placebo-controlled study. Two-hundred and eight volunteers, 96 therapy-naive with CD4 cell count >500x10(6)/l (group A) and 112 with CD4 cell count of 200-500x10(6)/l (group B, 51 out of 112 on treatment with one or two nucleoside analogues), received monthly injections of rgp160 IIIB vaccine or placebo for the first 6 months of the study; booster immunizations with rgp160 MN or placebo were given at times 15, 18, and 21 months. Safety and immunogenicity data were obtained and measurements of CD4 cell count, plasma viral RNA, and proviral DNA were performed. Clinical outcome was recorded for the 24 months of study. The vaccine was safe and well tolerated. Despite the induction of new rgp160-specific lymphoproliferative responses and the presence of positive delayed type hypersensitivity skin tests to rgp160 at the end of the 24 month study, no effect on the natural history of HIV infection was detected. Within 24 months, AIDS-defining illnesses had occurred in 19 of the vaccinated volunteers and in 18 of the placebo recipients. Persons with higher plasma viral RNA levels and higher proviral DNA had a more rapid decline in CD4 cell count when compared to persons with lower values. Vaccine did not alter viral RNA or proviral DNA levels. There was no clinical benefit to therapeutic immunizations with rgp160, despite the induction of new lymphoproliferative responses.
    AIDS 09/1999; 13(12):1461-8. · 6.56 Impact Factor
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    ABSTRACT: Vaccinia viruses defective in the essential gene coding for the enzyme uracil DNA glycosylase (UDG) do not undergo DNA replication and do not express late genes in wild-type cells. A UDG-deficient vaccinia virus vector carrying the tick-borne encephalitis (TBE) virus prM/E gene, termed vD4-prME, was constructed, and its potential as a vaccine vector was evaluated. High-level expression of the prM/E antigens could be demonstrated in infected complementing cells, and moderate levels were found under noncomplementing conditions. The vD4-prME vector was used to vaccinate mice; animals receiving single vaccination doses as low as 10(4) PFU were fully protected against challenge with high doses of virulent TBE virus. Single vaccination doses of 10(3) PFU were sufficient to induce significant neutralizing antibody titers. With the corresponding replicating virus, doses at least 10-fold higher were needed to achieve protection. The data indicate that late gene expression of the vaccine vector is not required for successful vaccination; early vaccinia virus gene expression induces a potent protective immune response. The new vaccinia virus-based defective vectors are therefore promising live vaccines for prophylaxis and cancer immunotherapy.
    Journal of Virology 07/1999; 73(6):4536-42. · 4.65 Impact Factor
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    ABSTRACT: An application of a quantitative PCR-based method was developed for the detection of human parvovirus B19 DNA. The procedure was characterised according to guidelines for the validation of analytical procedures. Furthermore, the reliability was demonstrated by the correct quantitation of samples of an international collaborative study. This application might be useful for studies focussed on removal and/or inactivation procedures of human parvovirus B19 as well as for general screening purposes of biological materials.
    Biologicals 10/1998; 26(3):213-6. DOI:10.1006/biol.1998.0129 · 1.41 Impact Factor
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    ABSTRACT: A sensitive and reliable quantitative method based on the polymerase chain reaction (PCR) and the reverse transcription polymerase chain reaction (RT-PCR) to detect and quantify human immunodeficiency virus (HIV-1) and hepatitis B virus (HBV), respectively, was developed. The samples are co-processed together with two internal standards (calibrators). The amplicons are separated on denaturing polyacrylamide gels and co-detected and quantitated by laser induced fluorescence. HIV-1 and HBV containing biological samples, including samples from international test panels, were accurately quantitated. The procedure has proven to be a valuable tool in the quality control of biologicals such as plasma products and may serve to monitor disease progression and response to antiviral therapy.
    Archives of Virology 02/1997; 142(7):1297-306. DOI:10.1007/s007050050161 · 2.28 Impact Factor
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    ABSTRACT: An assay was developed to specifically quantitate concentrations as low as 50 fg/ml genomic DNA based on the amplification of repetitive sequences. Reliable results were obtained by using internal standard molecules which were coextracted, coamplified, and coanalyzed with the nucleic acids of interest. Amplification was performed by the polymerase chain reaction in the presence of fluorescent dye-labeled primers followed by quantitation of fluorescence derived from the reaction products after separation by PAGE. Based on the known amount of added internal standard molecules and the intensities of the fluorescence of the reaction products, the primary results of the assay were obtained as copies per milliliter of sample. These were converted into mass units of DNA by applying an experimentally determined conversion factor. Chicken DNA has been used as an example for genomic DNA, and the sequences amplified were CR1 repetitive elements. This type of assay may be applied in cases where a sensitive and precise quantitation of genomic DNA is required, such as in the quality control of biological products.
    Analytical Biochemistry 12/1996; 242(2):240-7. DOI:10.1006/abio.1996.0459 · 2.31 Impact Factor
  • J Eibl, N Barrett, T Hämmerle, F Dorner
    Biologicals 10/1996; 24(3):285-7. · 1.41 Impact Factor
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    ABSTRACT: No abstract
    Biologicals 09/1996; 24(3):285-287. DOI:10.1006/biol.1996.0036 · 1.41 Impact Factor
  • T Hämmerle, F G Falkner, F Dorner
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    ABSTRACT: A sensitive and reliable quantitative method has been developed for the detection and quantitation of hepatitis C virus (HCV) target sequences. In this procedure, termed "laser induced fluorescence PCR' (LIF-PCR), reverse transcriptase PCR (RT-PCR) is performed and the PCR products are detected and quantified by laser-induced fluorescence. Precise quantitation of the viral target sequences is accomplished by the use of two calibrators that are amplified by the same set of primers as the target template. A high degree of reliability is achieved by co-processing, co-amplification and co-detection of the calibrators, together with the nucleic acid to be determined. Genome equivalents of HCV containing biological samples, including samples from international test panels, were accurately quantitated with this procedure.
    Archives of Virology 02/1996; 141(11):2103-14. DOI:10.1007/BF01718218 · 2.28 Impact Factor

Publication Stats

199 Citations
40.93 Total Impact Points

Institutions

  • 2006
    • IST Austria
      Klosterneuberg, Lower Austria, Austria
  • 1999–2003
    • Pennington Biomedical Research Center
      Baton Rouge, Louisiana, United States