Inactivation of West Nile virus, vaccinia virus and viral surrogates for relevant and emergent viral pathogens in plasma-derived products.

BACKGROUND AND OBJECTIVES: Human plasma is the source of a wide variety of therapeutic proteins, yet it is also a potential source of viral contamination. Recent outbreaks of emergent viral pathogens, such as West Nile virus, and the use of live vaccinia virus as a vaccine have prompted a reassessment of the viral safety of plasma-derived products. The purpose of this study was to evaluate the efficacy of current viral inactivation methods for West Nile and vaccinia viruses and to reassess the use of model viruses to predict inactivation of similar viral pathogens. MATERIALS AND METHODS: Virus-spiked product intermediates were processed using a downscaled representation of various manufacturing procedures. Virus infectivity was measured before and after processing to determine virus inactivation. RESULTS: The results demonstrated effective inactivation of West Nile virus, vaccinia virus and a model virus, bovine viral diarrhoea virus, during pasteurization, solvent/detergent treatment and caprylate treatment. Caprylate provided rapid and effective inactivation of West Nile virus, vaccinia virus, duck hepatitis B virus and Sindbis virus. Inactivation of West Nile virus was similar to that of bovine viral diarrhoea virus. CONCLUSIONS: This study demonstrates that procedures used to inactivate enveloped viruses in manufacturing processes can achieve inactivation of West Nile virus and vaccinia virus. In addition, the data support the use of model viruses to predict the inactivation of similar emergent viral pathogens.

Evaluation of virus and prion reduction in a new intravenous immunoglobulin manufacturing process.

BACKGROUND AND OBJECTIVES: Minimizing the transmission risk of infectious diseases is of primary importance in the manufacture of products derived from human plasma. A novel chromatography-based intravenous immunoglobulin (IGIV) manufacturing process was developed and the reduction of virus and transmissible spongiform encephalopathies (TSE) during the manufacturing process was assessed. Mechanistically distinct steps that could affect virus reduction were identified, and the robustness of virus reduction over the range of process conditions was determined. MATERIALS AND METHODS: Virus and TSE reduction by processing steps were assessed using a scaled-down version of the IGIV manufacturing process. RESULTS: Virus and TSE reduction at manufacturing process set points were well within safety standards. Robustness studies verified that the reproducibility of virus reduction was maintained at or beyond operating parameter extremes. Virus reduction across two combined manufacturing steps was lower than the sum of virus-reduction values across the individual steps, indicating mechanistic similarity of the two steps with respect to virus reduction. Only reduction from mechanistically distinct steps was claimed. CONCLUSIONS: This comprehensive approach to pathogen safety provides the new immunoglobulin manufacturing process with a detailed, yet realistic, assessment of the risk of transmission of infectious pathogens.

The HTLV receptor is a widely expressed protein.

The receptor for human T-cell leukemia virus type 1 (HTLV-1) was found to be expressed on a broad range of cell lines derived from multiple species. Receptor expression was assessed using human immunodeficiency virus type 1 particles, pseudotyped with the HTLV-1 envelope glycoprotein, and expressing luciferase under the control of an SV40 enhancer and promoter. Infection by pseudotyped virus was blocked with neutralizing antibodies to HTLV-1, and infection was dependent on the presence of the cleavage and fusogenic sequences in the envelope protein precursor. Trypsin treatment of susceptible target lymphocytes reduced entry. Entry was partially resistant to ammonium chloride.

The tax protein of human T-cell leukemia virus type 1 mediates the transactivation of the c-sis/platelet-derived growth factor-B promoter through interactions with the zinc finger transcription factors Sp1 and NGFI-A/Egr-1.

Transcriptional up-regulation of the c-sis/platelet-derived growth factor-B (PDGF-B) proto-oncogene by the Tax protein of human T-cell leukemia virus type 1 has been implicated as one possible mechanism of cellular transformation by human T-cell leukemia virus type 1. In previous work, we identified an essential site in the c-sis/PDGF-B promoter, Tax-responsive element 1 (TRE1), necessary for transactivation by Tax. We also identified Sp1, Sp3, and NGFI-A/Egr-1 as the primary nuclear transcription factors binding to TRE1 which mediate Tax responsiveness. In the present work, we have investigated the mechanism(s) whereby Tax transactivates the c-sis/PDGF-B proto-oncogene. In vitro transcription assays showed that Tax was able to significantly increase the transcriptional activity of a template containing the -257 to +74 region of the c-sis/PDGF-B promoter. Electrophoretic mobility shift assay analysis showed that Tax increased the DNA binding activity of both Sp1 and NGFI-A/Egr-1 using a TRE1 probe. Analysis of Tax mutants showed that two mutants, IEXC29S and IEXL320G, were unable to significantly transactivate the c-sis/PDGF-B promoter. Finally, co-immunoprecipitation analysis revealed that Tax is able to stably bind to both Sp1 and NGFI-A/Egr-1. Interestingly, co-immunoprecipitation analysis also revealed that Tax mutant IEXC29S is unable to interact with NGFI-A/Egr-1, whereas Tax mutant IEXL320G is able to interact with NGFI-A/Egr-1.

c-sis/PDGF-B promoter transactivation by the Yax protein of human T-cell leukemia virus type 1.

The human c-sis proto-oncogene promoter is transactivated by the human T-cell leukemia virus type 1 Tax protein in human Jurkat T-cells. Transactivation was >7-fold in Jurkat cells stably expressing the Tax protein (Jurkat-Tax) than in Jurkat E6.1 cells and was further enhanced in Jurkat-Tax cells stimulated with 12-O-tetradecanoylphorbol-13-acetate and the calcium ionophore, ionomycin. Deletion analysis showed that a 167-base pair promoter fragment retained full Tax responsiveness. Insertion of this minimal Tax-responsive region into a heterologous, minimal promoter resulted in approximately a 7-fold increase of transcriptional activation in the presence of Tax. Linker-scanning insertion analysis of this region identified Tax-responsive elements at nucleotides -64 to -45 (TRE1) and -34 to -15 (TATA box region). TRE1 contains a consensus binding site for the Sp family of transcription factors. The TATA box region corresponds to the TATA box and its 3'-neighboring sequence. Gel-shift and antibody supershift analysis of TRE1-binding proteins in unstimulated Jurkat E6.1 and Jurkat-Tax nuclear extracts identified Sp1 and Sp3 as the main TRE1 binding factors. Nuclear extracts from stimulated Jurkat E6.1 and Jurkat-Tax cells identified an additional TRE1 binding factor, Egr-1. These studies define a novel mechanism whereby Tax transactivates the c-sis promoter.