Production of parvovirus B19 vaccine in insect cells co-infected with double baculoviruses

Recombinant human parvovirus B19 virus-like particles (VLPs), a candidate vaccine, were produced using the insect cell (Sf-9)-baculovirus (AcNPV) expression system. The synthesis and assembly of the particles in Sf-9 cells are directed by double infections with one recombinant virus (bacVP1) expressing the parvovirus minor viral protein VP1 and a second virus (bacVP2) expressing the major viral protein VP2. Previous animal studies demonstrated that the polypeptide composition of the VLPs strongly affects the elicitation of virus neutralizing antibodies. The key factor controlling the production of an immunologically potent product in bioreactors was identified to be the multiplicity of infection (MOI) of bacVP1 and bacVP2 used for infection. A probabilistic model, which correlates well with the experimental results, was employed to facilitate the selection of MOIs and to provide a better understanding of the baculovirus co-infection process. A novel production process based on secondary infections was developed to ensure product consistency and to simplify large-scale logistics. The effects of other critical process parameters, such as temperature, dissolved oxygen concentration, lactate concentration, cell concentration at infection, and harvest time, were also investigated. (c) 1996 John Wiley & Sons, Inc.     

A novel nuclear receptor superfamily member in Xenopus that associates with RXR, and shares extensive sequence similarity to the mammalian vitamin D3 receptor.

We report the isolation of xONR1, a novel member of the nuclear receptor superfamily from Xenopus laevis. xONR1 shares a high degree of amino acid sequence identity with the mammalian receptor for 1 alpha, 25-dihydroxy vitamin D3, particularly within the DNA-binding domain, although it does not bind this ligand. xONR1 DNA binding is stimulated by association with retinoid X receptor gamma (RXR gamma).     

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.     

Extracellular pH determines the rate of Ca2+ entry into Madin-Darby canine kidney-focus cells

We investigated the relationship between intracellular Ca²⁺ and pH homeostasis in Madin-Darby canine kidney-focus (MDCK-F) cells, a cell line exhibiting spontaneous oscillations of intracellular Ca²⁺ concentration (Ca _i ²⁺ ). Ca _i ²⁺ and intracellular pH (pH _i ) were measured with the fluorescent dyes Fura-2 and BCECF by means of video imaging techniques. Ca²⁺ influx from the extracellular space into the cell was determined with the Mn²⁺ quenching technique. Cells were superfused with HEPES-buffered solutions. Under control conditions (pH 7.2), spontaneous Ca _i ²⁺ oscillations were observed in virtually all cells investigated. Successive alkalinization and acidification of the cytoplasm induced by an ammonia ion prepulse had no apparent effect on Ca _i ²⁺ oscillations. On the contrary, changes of extracellular pH value strongly affected Ca _i ²⁺ oscillations. Extracellular alkalinization to pH 7.6 completely suppressed oscillations, whereas extracellular acidification to pH 6.8 decreased their frequency by 40%. Under the same conditions, the respective pH _i changes were less than 0. 1 pH units. However, experiments with the Mn²⁺ quenching technique revealed that extracellular alkalinization significantly reduced Ca²⁺ entry from the extracellular space. Large increases of Ca _i ²⁺ triggered by the blocker of the cytoplasmic Ca²⁺-ATPase, thapsigargin, had no effect on pH _i We conclude: intracellular Ca²⁺ homeostasis in MDCK-F cells is pH dependent. pH controls Ca²⁺ homeostasis mainly by effects on the level of Ca²⁺ entry across the plasma membrane. On the contrary, the intracellular pH value seems to be insensitive to rapid changes of Ca _i ²⁺ .The project was supported by the Deutsche Forschungsgemeinschaft, SFB-176 (A6) and by the Jubilämusstiftung of the University of Würzburg.The authors gratefully acknowledge the valuable discussions with Drs. M.J. Berridge, M. Carew, I. Davidson, G. Law and B. Somasundraman. We are grateful to Applied Imaging for financial and technical support and to the Medical Research Council for financial support.     

Animal-derived antigenic variants of foot-and-mouth disease virus type A12 have low affinity for cells in culture.

We recently have shown that binding of foot-and-mouth disease virus (FMDV) to cells in culture requires an arginine-glycine-aspartic acid (RGD) sequence in the G-H loop of the capsid protein VP1 (P. W. Mason, E. Rieder, and B. Baxt, Proc. Natl. Acad. Sci. USA 91:1932-1936, 1994). In this report, we show that FMDV type A12 viruses found in infected bovine tongue tissue (BTT) differ from their tissue culture-grown derivatives at amino acid residues near the RGD. Viruses genetically engineered to contain VP1 sequences found in animal tissue (BTT viruses) were antigenically different from their tissue culture derivatives and bound to BHK cells more poorly than did the tissue culture-adapted viruses. Passage of the genetically engineered BTT viruses in BHK cells resulted in the rapid selection of variants with cell-binding properties, antigenic characteristics, and sequences typical of tissue culture-adapted viruses. These data indicate that residues near the RGD are critical for cell binding and that interpretations of antigenic variation of FMDV can be affected by virus cultivation in vitro.     

Proper maturation of the Japanese encephalitis virus envelope glycoprotein requires cosynthesis with the premembrane protein.

The role of the Japanese encephalitis virus (JEV) premembrane (prM) protein in maturation of the envelope (E) glycoprotein was evaluated by using recombinant vaccinia viruses encoding E in the presence (vP829) or absence (vP658) of prM. Immunofluorescence analyses showed that E appeared to be localized in the endoplasmic reticulum of cells infected with JEV, vP829, or vP658. However, reactivity with monoclonal antibodies and behavior in Triton X-114 indicated that E produced in the absence of prM behaved abnormally. Furthermore, E produced in the presence of prM by recombinant vaccinia viruses could be incorporated into flavivirus pseudotypes, whereas E synthesized in the absence of prM could not. These results demonstrate that cosynthesis of prM is required for proper folding, membrane association, and assembly of the flavivirus E protein.     

Constructed floating wetlands: a “safe-to-fail” study with multi-sector participation

The Duwamish River Floating Wetlands project designed, built, and deployed constructed floating wetlands in the estuary of the urban Duwamish River in Seattle, Washington, during the 2019 and 2020 outmigration seasons for juvenile salmon. Using a “safe-to-fail” methodology and adaptive management strategies, these innovative floating wetland prototypes were custom designed to provide the native plants, invertebrates and slow water habitat that juvenile salmon require during their transition from fresh to salt water, and were monitored for these outcomes. This paper will provide insight into the prototype designs, adaptive management strategies and plant performance, and unique public-private-academic-community partnerships that supported 2 years of design and research.