The primary production of an infectious recombinant Herpes Simplex Virus vaccine

The production and extracellular release of a recombinant Herpes Simplex Virus (type 2) from monolayers of infected complementing Vero cells (CR2) are addressed. Growth and virus production conditions are identified that provide adequate virus titers with cell seeding densities and viral multiplicities of infection that could be reasonably handled in manufacturing. Harvesting by sonication of cell monolayers is shown to give the highest recovery of infectious virus (to 2.5 x 10(6) pfu/mL) but leads to process stream contamination by cellular proteins through the rupturing of cells (to 28 pg protein/pfu). By comparison, freeze-thaw cycles and osmotic rupture by hypotonic saline or glycerol shock procedures yield only low virus recovery (typically <10% of that by sonication), and are accompanied by yet higher levels of protein contamination (up to 30-fold higher pg protein/pfu). Addition of the polyanionic polymers, heparin or dextran sulphate to a harvest using either hypotonic saline, glycerol shock or isotonic phosphate buffered saline increased the yield of infectious virus in the supernatant. By contrast, addition of polycationic poly-L-lysine resulted in negligible increase in the supernatant virus titer. The highest virus titers (4.7 x 10(7) pfu/mL) were achieved following treatment of roller bottle cultured cells displaying a high cytopathic effect with heparin at 50 microg/mL for at least 3 h post harvest. This procedure also gave the lowest levels of protein contamination (<2 pg protein/pfu). The fivefold lower yield of infectious virus from cultures displaying a low cytopathic effect (<70% CPE) indicates the importance of cell physiological state at harvest.     

Hydroxyapatite chromatography of phage-display virions

Hydroxyapatite column chromatography can be used to purify filamentous bacteriophage--the phage most commonly used for phage display. Virions that have been partially purified from culture supernatant by two cycles of precipitation in 2% polyethylene glycol are adsorbed onto the matrix at a density of at least 7.6 x 10(13) virions (about 3 mg) per milliliter of packed bed volume in phosphate-buffered saline (PBS; 0.15 M NaCl, 5 mM NaH2PO4, pH-adjusted to 7.0 with NaOH). The matrix is washed successively with wash buffer I(150 mM NaCl, 125 mM phosphate, pH 7.0), wash buffer II (2.55 M NaCl, 125 mM phosphate, pH 7.0), and wash buffer I; after which virions are desorbed in desorption buffer (150 mM NaCl, 200 mM phosphate, pH 7.0), and the matrix is stripped with stripping buffer (150 mM NaCl, 1 Mphosphate, pH 7.0). About half of the applied virions are recovered in desorption buffer. Western blot analysis shows that they have undetectable levels of host-derived protein contaminants that are present in the input virions and in virions purified by CsCl equilibrium density gradient centrifugation--the method most commonly used to prepare virions in high purity. Hydroxyapatite chromatography is thus an attractive alternative method for purifying filamentous virions, particularly when the scale is too large for ultracentrifugation to be practical.     

Isolation of lactoperoxidase, lactoferrin, alpha-lactalbumin, beta-lactoglobulin B and beta-lactoglobulin A from bovine rennet whey using ion exchange chromatography

A mild and rapid method is described for isolating various milk proteins from bovine rennet whey. beta-Lactoglobulin from bovine rennet whey was easily adsorbed on and desorbed from a weak anion exchanger, diethylaminoethyl-Toyopearl. However, alpha-lactalbumin could not be adsorbed onto the resin. alpha-Lactalbumin and beta-lactoglobulin from rennet whey could also be adsorbed and separated using a strong anion exchanger, quaternary aminoethyl-Toyopearl. The rennet whey was passed through a strong cation exchanger, sulphopropyl-Toyopearl, to separate lactoperoxidase and lactoferrin. alpha-Lactalbumin and beta-lactoglobulin were adsorbed onto quaternary aminoethyl-Toyopearl. alpha-Lactalbumin was eluted using a linear (0-0.15 M) concentration gradient of NaCl in 0.05 M Tris-HCl buffer (pH 8.5). Subsequently, beta-lactoglobulin B and beta-lactoglobulin A were eluted from the column with 0.05 M Tris-HCl (pH 6.8), using a linear (0.1-0.25 M) concentration gradient of NaCl. The yields were 1260 mg alpha-lactalbumin, 1290 mg beta-lactoglobulin B and 2280 mg beta-lactoglobulin A from 1 l rennet whey.     

Lagenin, a novel ribosome-inactivating protein with ribonucleolytic activity from bottle gourd (Lagenaria siceraria) seeds

The seeds of Lagenaria siceraria (Family Cucurbitaceae) were extracted with water and the extract was lyophilized. The lyophilized extract was chromatographed on a DEAE-cellulose column in 10 mM Tris-HCl buffer (pH 7.2). The unadsorbed fraction was applied to an Affi-gel Blue gel column previously equilibrated with the same buffer. After removal of unadsorbed materials, the adsorbed proteins were eluted with 1.5 M NaCl in the Tris-HCl buffer. After dialysis the adsorbed fraction was loaded on a CM-Sepharose CL-6B column which had been equilibrated with and was eluted with the same buffer. After elution of unadsorbed proteins, the column was eluted with a gradient of 0-1 M NaCl in 10 mM Tris-HCl buffer (pH 7.2). The fraction eluting at about 0.55 M NaCl, which represented pure ribosome inactivating protein (RIP), inhibited cell-free translation in a rabbit reticulocyte system with an IC50 of 0.21 nM and exerted ribonuclease activity on yeast tRNA with an activity of 45 U/mg. The RIP was designated lagenin. It possessed a molecular weight of 20 kDa, smaller than the range of 26-32 kDa reported for other RIPs. The N-terminal sequence of lagenin exhibited a lesser extent of similarity to those of other Cucurbitaceae RIPs, characterized by a deletion of the first three amino acid residues and a replacement of the 4th (Phe), 17th (Phe), 18th (Ile) and 22nd (Arg) residues which are invariant in other RIPs.     

A rapid and gentle method for the salt extraction of chromatin core histones H2A,H2B,H3 and H4 from rat liver nuclei

A complex of histones H2A, H2B, H3 and H4 has been isolated from purified rat liver nuclei by a method which is both gentle and rapid. Nuclei were homogenised in 0.25 M sucrose and the residual nuclear material obtained after centrifligation was adsorbed on calcium phosphate gel. After removing histone H1 from the adsorbed material by washing with 1M NaCl in 25 mM sodium phosphate buffer, pH 6.0, histones H2A, H2B, H3 and H4 were eluted together, with 2 M NaCl in 25 mM sodium phosphate buffer, pH 7.0. The core histones so obtained migrated as a single sharp band on polyacrylamide gel electrophoresis under non-denaturing conditions. Fractionation of the freshly prepared core histones on a Sephadex G-100 column yielded two major protein peaks. The peak having the larger elution volume contained histones H2A and H2B in equal amounts while the peak with the smaller elution volume contained all the four histones. Histones H3 and H4 were present in larger proportions in the second peak.     

A multiple extraction--centrifugation method for the recovery of viruses from waste water treatment plant effluents and sludges

Absorption of 14C-labelled poliovirus-2 to sedimentable solids of primary sludge samples collected from a secondary treatment facility during a 6-month period averaged 94%; for anaerobically digested sludge, 99%. The extent of virus adsorption was influenced by the amount of solids. Maximal adsorption occurred at or above 0.5% solids with sludge diluted with deionized water and above 1.5% solids when diluted with the respective particle-free sludge supernatants. A Tris-HCl buffer containing NaCl, glycerol, and serum was found to efficiently elute poliovirus-2 from primary sludge solids. By means of re-extraction and concentration by centrifugation (the TEC procedure), the average recoveries of poliovirus-2 were 92-94% based upon either infectivity or radioactivity analyses. Similarly, recoveries were 90-92% for poliovirus-2 in digested sludge. Maximum elution was dependent upon all four TEC buffer components and the restriction of solids to less than or equal to 1.0%. The procedure was found to be more efficient than glycine-NaOH and Freon procedures or elution with beef embryo extract. As adapted for effluents the procedure increased the yield and improved the consistency of virus recovery. The arithmetic mean titers and obtained during a monitoring study for primary and digested sludge were 4.2 X 10(5) and 5.1 X 10(3) plaque-forming units (pfu)/L; for primary, secondary, and final effluents 2.3 X 10(5), 4.7 X 10(3), and 4.7 X 10(2) pfu/L, respectively.     

DNA-binding proteins from Novikoff hepatoma cells.

In 0.05 M NaCl, 6-8% of the total soluble proteins from Novikoff hepatoma cells bind rapidly and reversibly to columns containing either heterologous or homologous DNA adsorbed to cellulose. These proteins can be eluted by buffer containing 2.0 M NaCl. 0.5-1% of the total protein exhibits a 7-17-fold preference for rat DNA over Escherichia coli DNA. 1-1.5% of the proteins bind DNA so strongly that elution cannot be effected by 4.0 M NaCl but can be accomplished by deoxyribonuclease I treatment of the columns. DNA-binding proteins eluted by 2.0 M NaCl were labeled with 125I or 131I and characterized by sodium dodecylsulfate-polyacrylamide gel electrophoresis and isoelectric focusing. These experiments indicate that DNA-binding proteins represent a discrete subset of the total soluble protein. Many similarities were noted between the major components of the homologous and heterologous DNA-binding fractions.     

Concentration of Enteroviruses on Membrane Filters

Enteroviruses can be made to adsorb or to pass through membrane filters by manipulation of the suspending medium. Salts facilitate virus adsorption, but membrane-coating components (MCC) interfere. Because cells release MCC into the culture medium during viral growth, MCC must be removed before virus can be adsorbed to membranes. Adsorbed virus can be eluted with diluents containing MCC (cell extracts or serum) or agents that reduce surface tension (sodium lauryl sulfate). By membrane adsorption and elution, enteroviruses can be readily concentrated and quantitatively recovered from crude virus harvests.     

Affinity chromatography of cysteine-containing histone.

Affinity chromatography based on the reaction between SH groups in protein and +HgC6H4CO groups in the p-mercuribenzoylaminoethyl derivative of Sepharose 4B was examined with a crude preparation of calf thymus cysteine-containing histone. Adsorption of the histone onto the column by specific coupling was found to be optimal in 0.1 M citrate buffer, pH 5.5, containing 5M urea to prevent any aggregation of histones and their non-specific adsorption onto the column, and elution from the column was successfully performed by cleavage of the resulting S-Hg bond with urea-buffer solution containing 0.05 M 2-mercaptoethanol. Under these conditions both the adsorption and elution were quantitative; no adsorption was observed when either SH-blocked histone or unsubstituted Sepharose was used. The cysteine-containing histone thus recovered, after further purification by Bio-Gel P-60 chromatography to remove some cysteine-containing nonhistone proteins contaminating the starting material, showed a single band on polyacrylamide gel electrophoresis and an amino acid composition agreeing with the known sequence of this histone.     

Purification of several bacteriolytic enzymes by affinity chromatography on lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with sepharose.

Using lysozyme-lysate of Micrococcus lysodeikticus cell wall coupled with Sepharose, several bacteriolytic enzymes were purified from crude preparations of animal and microbial origin. Quail egg-white, human milk and salivary lysozymes [EC 3.2.1.17] were adsorbed onto the adsorbent at pH 5-7 and eluted with 2M NaCl at pH 10. By means of these treatments, lysozymes were purified 20-250 fold with activity recoveries of 60-80%, and the quail lysozyme thus purified was shown to be discelectrophoretically homogeneous. Some bacteriolytic enzymes of microbial origin were also highly purified by using this affinity adsorbent. A bacterial lysozyme from Bacillus sp. ML-208 showed high affinity for the ligand and was not eluted under the conditions mentioned above, but was recovered by elution with 2M guanidine-HCl at pH 5.8, resulting in a 500-fold increase in the specific activity. A Pseudomonas-lytic enzyme from Streptomyces sp. P-51 was easily released from the adsorbent by elution with 0.5M NaCl at pH 5.0. A staphylolytic F2 enzyme from S. griseus S-35 and a chitinase [EC 3.2.1.14] from yam, both of which were completely inert toward M. lysodeikticus cell wall, passed through the adsorbent column. A modified ligand, in which muramic acid and glucosamine residues were N,O-acetylated, failed to adsorb any of these animal and bacterial lysozymes. Some of the enzymatic properties and bacteriolytic action spectra of these purified enzymes are also described in this paper in comparison with those of hen egg-white lysozyme.     

Incorporation of Radioactive Seleno-(75Se)-Methionine into Mumps Virus

Mumps virus was grown in embryonated chicken eggs in the presence of radioactive seleno-(⁷⁵Se)-methionine. Virus in the allantoic and amniotic fluids was concentrated in a sucrose density gradient, and a peak of viral material coincided with a significant peak of ⁷⁵Se-radioactivity. The radioactivity was acid-insoluble and remained associated with the virus after purification by erythrocyte adsorption and elution and centrifugation on a second sucrose density gradient. After amino-acid hydrolysis of the radioactive virus, only ⁷⁵Se-methionine was recovered by chromatographic analysis. These results demonstrate that the radioactive ⁷⁵Se-methionine was incorporated into protein of infectious mumps virus.     

Isolation of a novel agglutinin with complex carbohydrate binding specificity from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji.

A hemagglutinin, with a molecular weight of 30,000 and expressing hemagglutinating activity which could not be inhibited by simple sugars and glycoproteins, was isolated from fresh fruiting bodies of the edible mushroom Lyophyllum shimeiji. The protein was adsorbed on CM-Sepharose even in 20 mM ammonium acetate (pH 5.5) containing 1 M NaCl and was desorbed by 20 mM ammonium bicarbonate (pH 9). The hemagglutinating activity was subsequently adsorbed on Mono S in 20 mM ammonium acetate (pH 5.5) and was desorbed by a linear gradient of 0.2-0.5 M NaCl in ammonium acetate buffer. The hemagglutinin exhibited a novel N-terminal sequence not found in any lectin and hemagglutinin reported so far. It was devoid of antifungal activity.     

Efficient inclusion body processing using chemical extraction and high gradient magnetic fishing

In this study we introduce a radical new approach for the recovery of proteins expressed in the form of inclusion bodies, involving (i) chemical extraction from the host cells, (ii) adsorptive capture of the target protein onto small magnetic adsorbents, and (iii) subsequent rapid collection of the product-loaded supports with the aid of high gradient magnetic fields. The manufacture and testing of two types of micron-sized nonporous superparamagnetic metal chelator particles derivatized with iminodiacetic acid is described. In small-scale adsorption studies conducted with a hexahistidine tagged form of the L1 coat protein of human papillomavirus type 16 dissolved in 8 M urea-phosphate buffer, the best binding performance (Q(max) = 58 mg g(-1) and K(d) approximately 0.08 microM) was exhibited by Cu(2+)-charged type II support materials. Equilibrium adsorption of L1 to these nonporous supports was achieved very rapidly (<300 s), and approximately 90% of the tightly bound L1 could be desorbed in just one elution step by including >100 mM imidazole in the equilibration buffer. The influence of feedstock complexity on L1 adsorption to the Cu(2+)-charged type II magnetic chelators was studied using various dilutions of four crude chemical E. coli cell extracts containing denatured L1 protein. Undiminished L1 adsorption to these adsorbents (relative to the 8 M urea-phosphate buffer case) was observed with the least complex of these feed materials, i.e., a partially clarified (12 g dry weight L(-1)) and spermine-treated chemical cell extract (feedstock B). Efficient recovery of L1 from feed B was demonstrated at a 60-fold increased scale using the high gradient magnetic fishing (HGMF) system to collect loaded Cu(2+)-chelator particles following batch adsorption of L1. Over 70% of the initial L1 present was recovered within the HGMF rig in a highly clarified form in two batch elution cycles with an overall purification factor of approximately 10.     

Adsorption chromatography of nucleic acids on silicone-coated porous glass

Bovine liver tRNA was adsorbed on silicone-coated porous glass in 5 M NaCl, 10 mM Tris-HCl (pH 7.6) and fractionated by elution with decreasing NaCl concentrations. tRNAPro, tRNAVal, tRNAIle, tRNAThr, tRNASer, and tRNAPhe were eluted in this order. tRNA which had been digested with ribonuclease A was not adsorbed. Q beta RNA (adsorbed onto the glass in 5 M NaCl) was eluted with 1.5 M NaCl. RNA species in a crude rRNA fraction from Escherichia coli were separated into tRNA, 5S rRNA, and high molecular weight rRNA on siliconized porous glass. A half of calf thymus DNA was adsorbed on the glass in 5 M NaCl and the residual part passed through the column. The CD spectra showed that DNA and tRNA took the C-form and the A-form in 5 M NaCl, respectively. Therefore, the discrepancies of behavior of the DNA and RNA on siliconized porous glass may be related to the occurrence of these forms. The recovery of these nucleic acids from the column was 83-100%. Adsorption chromatography on siliconized porous glass may be a useful method for the separation of tRNA, rRNA, and mRNA.     

Hydrophobic interaction chromatography of the rabbit uterine progesterone receptor

The chromatographic behavior of the rabbit uterine progesterone receptor interaction on several different hydrophobic matrices was characterized. Receptor, prepared in 0.6 M NaCl, exhibited a progressive retardation of elution, followed by retention, on a series of alkyl agarose columns as the length of the alkyl chain [(CH2)nH-] increased (n = 0-10), reflecting the presence of hydrophobic regions on the protein. Adsorption did not occur directly at the steroid binding site of the molecule and did not require activation to the DNA-binding form. Elution could be achieved by a decrease in the ionic strength of the buffer or the addition of glycerol, resulting in partial purification of receptor. Receptor bound tightly to phenyl agarose, although elution of the receptor under mild conditions (decreasing salt gradient, increasing glycerol gradient) resulted in poor yield and only modest purification. Passage of the non-activated progesterone receptor over Reactive Blue Sepharose effectively removed albumin, presumably by a hydrophobic interaction, although receptor was not retained. In the activated form, approximately 25% of receptor was bound to Reactive Blue Sepharose, reflecting an interaction of the Cibacron Blue dye with the polynucleotide binding site of the receptor. Hydrophobic chromatography may be an important adjunct to methods for purification of the progesterone receptor.     

Production of high titre disabled infectious single cycle (DISC) HSV from a microcarrier culture

Disabled Infectious Single Cycle (DISC) HSV-2 has been cultured in the complimentary cell line CR2 to provide high titre bulk material suitable for the purification of the virus as a live viral vaccine. CR2 cells are cultured on the microcarrier Cytodex-1 at 5 g l-1 in small scale (1 l) and larger scale (15 l) reactors. The cells are infected at an MOI of 0.01 pfu cell-1 and the culture harvested 60–72 h later. The infected cells are removed from the microcarriers by the addition of a hypotonic saline and the virus released by low-pressure disruption techniques. Virus titres achieved are compared to the standard roller bottle process. The resulting material is the starting point for the purification of the DISC-HSV virus. This revised version was published online in July 2006 with corrections to the Cover Date.     

Concentration and purification of enteroviruses by membrane chromatography.

A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery.     

Concentration and purification of enteroviruses by membrane chromatography.

A simple procedure for the concentration and partial purification of enteroviruses from tissue culture harvests is described. After removal of acid-precipitating components with a cationic detergent, the detergent and most membrane-coating components were removed by treatment with a cationic-exchange resin. The resin effluent was then acidified, and the virus was adsorbed to epoxy-fiberglass membranes. Virus was then eluted with pH 11.5 glycine-NaOH buffer. Since this eluate contains no orgcentrated simply by acidifying the eluate and passing it through a smaller membrane than that used for the first concentration. As high as 500-fold concentrations can be achieved, with a high efficiency of recovery.     

Certain characteristics of isolation of human myelomic IgG of different subclasses and their distribution according to the allotypes, subclasses and types of light chain]

A combination of the methods of preparative electrophoresis in agar gel and of the ion-exchange chromatography on DE-32 cellulose permitted to obtain 32 immunochemically pure human myelomic IgG. The proteins of the first three subclasses were obtained by elution in the 0.01 phosphate buffer at pH 7.6. IgG4 was eluted with the increase of the gradient to 1 M NaCl in the phosphate buffer. Of the 32 human myelomic IgG 26 represented IgG1,4--IgG2, 1--IgG3, and 1--IgG4. Among the 26 IgG1 11 were of the Gm(a) allotype, and 15 proteins had the Gm(f) determinant; one IgG2 protein was Gm(n+) and 3--Gm(n-). One IgG3 protein was referred to the Gm(b) variant. The majority of the IgG proteins of the subclass I had chi-type of the L-chains, and the chi: lambda ratio constituted 2.71.