A new liquid intravenous immunoglobulin with three dedicated virus reduction steps: virus and prion reduction capacity.

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: Virus and prion removal capacities were assessed with down-scale spiking experiments, validated for equivalence to the large-scale process. RESULTS: 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. CONCLUSIONS: Introducing three dedicated virus-clearance steps in the manufacturing process of immunoglobulins from human plasma provides high margins of safety.

Quantitation of viral DNA by real-time PCR applying duplex amplification, internal standardization, and two-color fluorescence detection.

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.

A systematic and quantitative analysis of PCR template contamination.

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.

Highly efficient induction of protective immunity by a vaccinia virus vector defective in late gene expression.

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.

Precise quantitation of human parvovirus B19 DNA in biological samples by PCR.

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.

A sensitive PCR assay system for the quantitation of viral genome equivalents: human immunodeficiency virus type 1 (HIV-1) and hepatitis B virus (HBV).

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.

Specific quantitation of genomic DNA in the femtogram range by amplification of repetitive sequences.

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.

A sensitive PCR assay system for the quantitation of viral genome equivalents: hepatitis C virus (HCV).

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.

Purification and characterization of poliovirus polypeptide 3CD, a proteinase and a precursor for RNA polymerase.

A cDNA clone encoding the 3CD proteinase (3CDpro) of poliovirus type 2 (Sabin), the precursor to proteinase 3Cpro and RNA polymerase 3Dpol, was expressed in bacteria by using a T7 expression system. Site-specific mutagenesis of the 3C/3D cleavage site was performed to generate active proteolytic precursors impaired in their ability to process themselves to 3Cpro and 3Dpol. Of these mutations, the exchange of the Thr residue at the P4 position of the 3C/3D cleavage site for a Lys residue (3CDpro T181K) resulted in a mutant polypeptide exhibiting the smallest amount of autoprocessing. This mutant was purified to 86% homogeneity and used for subsequent proteolytic studies. Purified 3CDproM (M designates the cleavage site mutant 3CDpro T181K) was capable of cleaving the P1 capsid precursor, a peptide representing the 2BC cleavage site, and the 2BC precursor polypeptide. Purified 3CDproM demonstrated the same detergent sensitivity in processing experiments with the capsid precursor as was observed by using P1 and crude extracts of poliovirus-infected HeLa cell lysates. Purified 3CDproM did not have any detectable RNA polymerase activity, whereas 3Dpol, separated from 3CDproM by gel filtration in the last step of purification, did. We conclude that 3CDproM can process both structural and nonstructural precursors of the poliovirus polyprotein and that it is active against a synthetic peptide substrate. Moreover, cleavage of 3CD to 3Dpol is needed to activate the 3D RNA polymerase.

Incorporation of azaglutamine residues into peptides synthesised by the ultra-high load solid (gel)-phase technique.

The solid (gel)-phase peptide synthesis of peptides, each containing an azaglutamine residue, has been examined. Procedures using various mono-, di- and tripeptide and carbazate fragments containing or relating to an azaglutamine (1) residue have been evaluated. N-Activation of the amino-terminus of a resin-bound peptide with bis-(2,4-dinitrophenyl)carbonate (2) yielded the terminal isocyanate species, which reacted with protected carbazates to give resin-bound protected peptides containing the aza-residue. By contrast, coupling of activated amino-acid derivates to the free amino-group of a resin-bound peptide with an aza-residue at the N-terminus was a slow and unsatisfactory process. It is concluded that the route yielding the best results involves the reaction of a protected amino-acyl carbazate to a resin-bound isocyanate-activated peptide.

Mutational analysis of the proposed FG loop of poliovirus proteinase 3C identifies amino acids that are necessary for 3CD cleavage and might be determinants of a function distinct from proteolytic activity.

Mutations were introduced into a cDNA clone of poliovirus resulting in single-amino-acid substitutions within the region of the proposed FG loop of proteinase 3C. RNAs were made by in vitro transcription with T7 RNA polymerase and used to transfect HeLa cells. Virus viability was assessed as indicated by cell lysis. In parallel, RNAs were translated in vitro by using a HeLa cell lysate, and the patterns of the processed poly-proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Replacement of Lys-78, Arg-79, and Glu-81 had apparently no effect on virus viability and on proteolytic processing. In contrast, virus viability was abolished by mutation of Phe-83, Arg-84, Asp-85, Ile-86, and Arg-87. With respect to substitution of Phe-83, Asp-85, and Arg-87, these effects correlated with impaired processing of the 3CD cleavage site, separating 3C and 3D, and, to a lesser extent, of the P1 precursor. Replacement of Arg-84 and Ile-86, on the other hand, did not alter the processing pattern. Thus, the lethal effects in these mutant genomes may not have been caused by impaired processing. A special case was the mutant of Lys-82-Gln. Virus recovered from cells transfected with RNA carrying this mutation always contained an A-to-G transition which resulted in the replacement of glutamine for arginine. Our data suggest that residues in the proposed FG loop of proteinase 3C influence 3CD cleavage and that they are determinants of a function unrelated to proteolytic processing.

Infection of HeLa cells with poliovirus results in modification of a complex that binds to the rRNA promoter.

In HeLa cells, RNA polymerase I (Pol I)-mediated transcription is severely inhibited soon after infection with poliovirus. We have developed a gel retardation assay to analyze DNA-protein complexes formed at the Pol I promoter. We show here that two complexes (A and C) formed by nuclear extracts from uninfected cells disappear after infection of cells with poliovirus. In contrast, a new, rapidly migrating complex (D) is formed in virus-infected cell extract. This change in the mobility of gel-retarded complexes correlates well with the kinetics of inhibition of rRNA transcription in virus-infected cells. Incubation of nuclear extracts from mock-infected cells with bacterially expressed, purified poliovirus protease 3C results in the disappearance of complexes A and C with concomitant generation of complex D. A partially purified transcription factor fraction derived from uninfected cells that contains complex A is able to restore Pol I transcription when added to virus-infected cell extracts, suggesting that this complex plays an important role in Pol I transcription. These results suggest that poliovirus proteinase 3C may have an important role in the shutoff of Pol I transcription in cells infected with poliovirus.

Poliovirus proteinase 3C converts an active form of transcription factor IIIC to an inactive form: a mechanism for inhibition of host cell polymerase III transcription by poliovirus.

In HeLa cells, RNA polymerase III (pol III)-mediated transcription is severely inhibited by poliovirus infection. This is due primarily to a reduction in the transcriptional activity of TFIIIC, a transcription factor which binds in a sequence specific manner to the internal promoter of pol III genes. Using gel retardation assays, we have shown previously that inhibition of pol III transcription by poliovirus is correlated with disappearance of a transcriptionally active form of TFIIIC (complex I) concomitant with the appearance of a faster mobility, transcriptionally inactive form of TFIIIC (complex III). We show here that a poliovirus with a point mutation in the proteinase 3C (3Cpro) region failed to produce complex III and is limited in its ability to inhibit pol III transcription compared with the wild-type virus. Incubation of purified 3Cpro, expressed in Escherichia coli, with transcriptionally active TFIIIC (complex I) in vitro resulted in generation of the transcriptionally inactive complex III form of TFIIIC. In an in vitro transcription assay, treatment of the complex I form of TFIIIC with 3Cpro almost completely inhibited pol III transcription. Finally expression of the 3Cpro gene in transfected HeLa cells resulted in significant inhibition of pol III-mediated transcription. The results presented here suggest that proteolysis of the transcriptionally active form of TFIIIC by poliovirus 3Cpro is a mechanism by which poliovirus inhibits host cell RNA pol III transcription.

Site-directed mutagenesis of the putative catalytic triad of poliovirus 3C proteinase.

Based on predictions of the structure of proteinase 3C of poliovirus, mutations have been made at residues that are supposed to constitute the catalytic triad. Wild-type and mutant 3C were expressed in Escherichia coli, purified to homogeneity, and characterized by the ability to cleave a synthetic peptide substrate or an in vitro translated polypeptide consisting of part of the polyprotein of poliovirus. Additionally, the ability of autocatalytic processing of a precursor harboring wild-type or mutant 3C sequences was tested. Single substitutions of the residues His-40, Glu-71, and Cys-147 by Tyr, Gln, and Ser, respectively, resulted in an inactive enzyme. Replacement of Asp-85 by Asn resulted in an enzyme that was as active as wild-type enzyme in trans cleavage assays but whose autoprocessing ability was impaired. Our results are consistent with the proposal that residues His-40, Glu-71, and Cys-147 constitute the catalytic triad of poliovirus 3C proteinase. Furthermore, residue Asp-85 is not required for proper proteolytic activity despite being highly conserved between different picornaviruses. This indicates that Asp-85 might be involved in a different function of 3C.

Simultaneous analysis of mitochondrial activity and DNA content in Ehrlich ascites tumor cells by dual parameter flow cytometry.

Ehrlich ascites tumor cells were permeabilized using low concentrations of digitonin, 8 micrograms/10(6) cells. Permeabilization was monitored by the assay of lactate dehydrogenase released into the incubation medium and of hexokinase partially bound to mitochondria. Integrity of the cellular organelles was unaffected as determined by assay of the mitochondrial enzyme glutamate dehydrogenase. Cells were stained with rhodamine 123 as a mitochondrial specific dye and propidium iodide/mithramycin as DNA specific dyes. The green fluorescence of bound rhodamine 123 versus red fluorescence of DNA in individual cells was analysed by dual parameter flow cytometry. Incubation of cells with inhibitors of mitochondrial energy metabolism, such as, potassium cyanide and carbonyl cyanide m-chlorophenylhydrazone abolished binding of rhodamine 123. Flow cytometric data allowed a correlation between cell position in the mitotic cycle with total mitochondrial activity. In addition, comparison of the characteristics of propidium iodide and ethidium bromide staining further elucidated the molecular basis of the staining with the positively-charged fluorescent dye rhodamine 123.

Activation of low Km hexokinases in purified hepatocytes by binding to mitochondria.

Hepatocytes were purified on a Percoll gradient. The cell membrane of these hepatocytes was disrupted by digitonin in the presence of albumin, glucose and physiological concentrations of monovalent and divalent cations. This treatment led to a separation between free and loosely structure-bound cytosolic enzymes which is not achieved by conventional subfractionation techniques. According to kinetic and immunological analyses, the free extractable cytosolic fraction contained high Km, hexokinase (glucokinase) and less than 10% of low Km hexokinases, while the hexokinase activity bound to the cell structures represented exclusively low Km isozymes. The total activity of the bound hexokinases was comparable to that observed in the supernate (approx. 1.0 U per g fresh weight). This activity decreased more than 10-fold upon desorption at higher digitonin concentrations. Such activation by binding, as well as inactivation by desorption, could also be demonstrated in intact hepatocytes correlated to different metabolic states, and also in vitro with isolated mitochondria and purified isozyme I. The binding of low Km hexokinases in hepatocytes was restricted to the mitochondrial fraction and there it was observed in the contact sites between the two mitochondrial boundary membranes. In view of these findings it appears that the binding-dissociation equilibrium of low Km hexokinases plays an important role in metabolic regulation of glucose uptake and glycogen synthesis in the liver and presumably in muscle.