Purification Of Influenza Virus Research Articles

Upcoming Chromatography Media for Cell Culture‐Derived Influenza Virus Purification

Upcoming Chromatography Media for Cell Culture‐Derived Influenza Virus Purification

An alternative method for preparation of pandemic influenza strain-specific antibody for vaccine potency determination

The traditional assay used to measure potency of inactivated influenza vaccines is a single-radial immunodiffusion (SRID) assay that utilizes an influenza strain-specific antibody to measure the content of virus hemagglutinin (HA) in the vaccine in comparison to a homologous HA reference antigen. Since timely preparation of potency reagents by regulatory authorities is challenging and always a potential bottleneck in influenza vaccine production, it is extremely important that additional approaches for reagent development be available, particularly in the event of an emerging pandemic influenza virus. An alternative method for preparation of strain-specific antibody that can be used for SRID potency assay is described. The approach does not require the presence or purification of influenza virus, and furthermore, is not limited by the success of the traditional technique of bromelain digestion and purification of virus HA. Multiple mammalian expression vectors, including plasmid and modified vaccinia virus Ankara (MVA) vectors expressing the HAs of two H5N1 influenza viruses and the HA of the recently emerging pandemic H1N1 (2009) virus, were developed. An immunization scheme was designed for the sequential immunization of animals by direct vector injection followed by protein booster immunization using influenza HA produced in vitro from MVA vector infection of cells in culture. Each HA antibody was highly specific as shown by hemagglutination inhibition assay and the ability to serve as a capture antibody in ELISA. Importantly, each H5N1 antibody and the pandemic H1N1 (2009) antibody preparation were suitable for use in SRID assays for determining the potency of pandemic influenza virus vaccines. The results demonstrate a feasible approach for addressing one of the potential bottlenecks in inactivated pandemic influenza vaccine production and are particularly important in light of the difficulties in preparation of potency reagent antibody for pandemic H1N1 (2009) virus vaccines.

Concentration and purification of influenza virus from allantoic fluid

A simple procedure which enables the concentration and purification of influenza virus, using an angular rotor, is described. Virus is concentrated over a sucrose step gradient. The same gradients are reused and volumes up to 4 liters are concentrated in 1 day. The concentrated virus is further purified by a simplified density-gradient technique. No host cell protein is detectable in the final product. The technique offers a broad application potential for concentrating and purifying other viruses.

The rapid concentration and purification of influenza virus from allantoic fluid

Influenza virus was quantitatively recovered from infectious allantoic fluid by precipitation with 8 percent (wt/vol) polyethylene glycol 6000 (PEG-6000). Virus concentrated by PEG-6000 treatment was rapidly purified by gel permeation chromatography through controlled pore glass beads. The purity of virus preparations achieved by this method, when judged by three independent criteria, was comparable to virus preparations purified by conventional density gradient procedures.

Purification of influenza virus with Arcton 113.

Arcton 113 has proved effective in the removal of host membrane fragments which cosediment with influenza virus during differential centrifugation. The virus appeared morphologically undamaged by the procedure and retained its infectivity, neuraminidase, and hemagglutinating activity. Pyrogenicity, however, was considerably reduced.

Purification of influenza viruses using disulphide-linked immunosorbents derived from rabbit antibody

Influenza viruses (A/Moscow/1019/65, A/England/344/68, PR/8-A/Hong kong/68) were specifically adsorbed from crude egg grown suspensions by their homologous disulphide-linked immunosorbents prepared from the γ-globulin of rabbits immunized with influenza virus from calf-kidney tissue cultures. In batch experiments 20 mg of the appropriate ‘non-avid’ immunosorbents (from 4–7 ml of serum taken shortly after one dose of antigen) adsorbed approximately 6 × 10 9, 3 × 10 9 and 3 × 10 10 egg-bit infective dose 50 (approximately 1 × 10 12, 1·3 × 10 12 and 1·9 × 10 12 total particles as indicated by haemagglutination assays) of A/Moscow/1019/65, A/England/344/68 and PR/8-A/Hong Kong/68 respectively; and about 50 per cent of infective virus was eluted with alkaline buffers of pH 11·3, 12·0 and 12·5 respectively. After filtration to remove a trace of immunosorbent the eluted virus suspensions contained 40–91,000 HA/mg protein. ‘Avid’ immunosorbents (prepared from serum taken later after booster injections) had greater capacities (approximately 2–4 times) for infective virus than ‘non-avid’ immunosorbents but only 3–18 per cent of virus could be eluted from them. Both ‘non-avid’ and ‘avid’ immunosorbents adsorbed heterologous viruses of the same or different serotypes to those used to prepare the immunosorbents provided there was some similarity in either the haemagglutinin or neuraminidase antigens. However, their capacities for heterologous viruses were reduced in comparison to those for homologous viruses. Using a column containing a mixture of immunosorbent (100 mg) and Sephadex G-15 approximately 3·5 × 10 12 total particles were adsorbed and 50 per cent eluted at each cycle at 5°C. Prior diafiltration of the crude virus suspensions was necessary to prevent column blockage but then the column could be recycled 10 times. The eluted virus suspension had 100,000 HA/mg protein and contained little or no egg or rabbit protein as judged by hypersensitivity reactions in suitably sensitised guinea-pigs.

Purification of influenza viruses using disulphide-linked immunosorbents derived from rabbit antibody

Purification of influenza viruses using disulphide-linked immunosorbents derived from rabbit antibody

Concentration and purification of influenza virus on insoluble polyelectrolytes.

A method for rapid concentration and purification of influenza virus by adsorption on and elution from an insoluble polyelectrolyte is described. To accomplish this task, influenza virus had to be rendered stable at pH 4 to 5, since viruses adsorb to the polyelectrolyte more efficiently at this pH range. A precipitate which forms in influenza harvests under acid conditions in the cold can be removed by ammonium sulfate at a concentration which traps the precipitate but not the virus. Thus, ammonium sulfate-treated influenza virus in allantoic fluid could be readily concentrated on the polyelectrolyte. Elution yielded a virus concentrate essentially free of nonviral proteins.

Concentration and Purification of Influenza Virus on Insoluble Polyelectrolytes

A method for rapid concentration and purification of influenza virus by adsorption on and elution from an insoluble polyelectrolyte is described. To accomplish this task, influenza virus had to be rendered stable at pH 4 to 5, since viruses adsorb to the polyelectrolyte more efficiently at this pH range. A precipitate which forms in influenza harvests under acid conditions in the cold can be removed by ammonium sulfate at a concentration which traps the precipitate but not the virus. Thus, ammonium sulfate-treated influenza virus in allantoic fluid could be readily concentrated on the polyelectrolyte. Elution yielded a virus concentrate essentially free of nonviral proteins.

Purification of influenza virus by phase separation and selective adsorption

Purification of influenza virus by phase separation and selective adsorption

Purification of influenza virus in the K-II zonal centrifuge.

VIRAL material used to prepare inactivated virus vaccines must be concentrated to provide sufficient antigenic mass to stimulate the vaccine's protective mechanisms, and purified to remove nonviral host material which may cause side reactions and sensitization to host (for example, egg) components. A continuous-flow zonal centrifuge, the K-II-B1,2, has been designed for use in large scale production of influenza vaccines. We have grown influenza virus in tissue culture and have concentrated and purified it in a single step process using this centrifuge.

Importance of the ionic form of ion exchangers for chromatography of macromolecular substances: Purification of influenza viruses

Importance of the ionic form of ion exchangers for chromatography of macromolecular substances: Purification of influenza viruses

DIRECT IDENTIFICATION OF INFLUENZA VIRUS IN THROAT WASHINGS BY MEANS OF CONCENTRATION AND MICRO-ASSAY.

SummaryA method of concentration and partial purification of influenza virus (A2) found in throat washings is described. A microtechnique is presented for identification of virus contained in the concentrates.The author wishes to thank Dr. Lajos Ccizmas, New York City Department of Health, for his generous gift of the Takatsy Plexiglas plates used in the micro-tests; Dr. Stephen Millian, Bureau of Laboratories, New York City Department of Health, for supplying the skim milk throat washings.The author is indebted to Dr. Benjamin Mandel, Public Health Research Institute of New York City, for valuable suggestions and encouragement.

A rapid specific method for the isolation of pneumococcal polysaccharide.

THE report by Gessler et al. 1 on the use of a completely substituted ethane (trichlorotrifluoro ethane, ‘Genetron 113’; obtained from the Division of Allied Dye and Chemical Corp., General Chemicals, Inc., New York) as a ‘deproteinizing’ agent in the purification of influenza virus, suggested to us that this solvent might have many uses. In particular, we were concerned with ‘deproteinizing’ bacterial cells with the consequent release of polysaccharides. In view of the observation that this solvent is not miscible with water and that pneumococcal poly-saccharide is soluble in water, the following experiment was performed.

Differentiation and Purification of Influenza Viruses by Adsorption on Aluminum Phosphate

Summary A study of the adsorption of five strains of influenza virus on aluminum phosphate has been presented. Each strain shows a characteristic adsorption pattern which can be used for rapid and efficient differentiation. This information has been applied to the separation of an artificial mixture of the PR8 and Lee strains. The method was applied to the purification of large batches of Melbourne strain by the use of supported columns. Eleven purified samples of virus harvested 14 hours after inoculation of 10-3 diluted seed virus gave an average log (ID50/g.N) of 15.2 with a range of 15.0 to 15.3. Criteria for the degree of purity of these samples have been presented. Within the limits of experimental error, the yield was quantitative. No significant inactivation of virus was encountered.

STUDIES ON THE PURIFICATION OF INFLUENZA VIRUS.

It has been well known that differential centrifugation by the use of ultracentrifuge is the most effective method for the purification of animal viruses. However, as the use of ultracentrifuge is so far fairly limited in this country, other methods for partially purifying viruses should be investigated more in detail. Adsorption and elution of viruses by means of adsorbents have been attempted by numerous workers for the partial purification of viruses. As adsorbents, resins (1) (2), clay (3), kaolin (4) (5) (6) as well as chicken red blood cells (7) (8) (9), in the case of viruses having hemagglutinins, have been employed. In recent years it has been shown that methyl alcohol at low temperature precipitates various proteins as well as viruses without denature or inactivation. And this fact has found wide application in the purification of various viruses (10) (11) (12) (13). The present work deals with the purification of influenza virus by methods in which adsorbents and precipitants are used or combined.

The Purification and Concentration of Influenza-Virus by Means of Alcohol Precipitation

Summary Experiments are reported in which ethyl and methyl alcohol were used to concentrate and partially purify the PR-8, Weiss and Lee strains of influenza-virus under carefully controlled conditions of alcohol-concentration, temperature and pH. Of the two, methanol was found to be superior since the optimal concentration is of broader range and less denaturation of viral protein is encountered than with ethanol. The data indicate that methanol-precipitation under controlled conditions produces no virus-denaturation nor loss of activity as judged by CCA activity, infectivity titers in chick-embryos or immunizing potency of killed vaccines for mice. On the contrary, based on the CCA activity of the starting material, yields of virus equal to or exceeding 100 per cent are obtained. The methanol-precipitation method is readily applicable to the concentration and purification of influenza-virus using either the bucket, angle head or Sharples continuous-flow types of centrifuges. The method is particularly valuable from the standpoint of producing vaccines on a large scale since it may be combined readily with the Sharples centrifuge method and thus effect great savings in man and machine hours. Preliminary experiments also show that the method may have wide application in the concentration and purification of other viral and rickettsial agents.

A SEDIMENTABLE COMPONENT OF ALLANTOIC FLUID AND ITS RELATIONSHIP TO INFLUENZA VIRUSES

Macromolecular material was isolated from normal allantoic fluid by a centrifugation procedure comparable to that currently employed for the concentration and purification of influenza viruses. The yield of material was found to vary with the age of the embryo, reaching a maximum average value after 14 days of incubation at 39 degrees C. of about 0.02 mg. per ml. of allantoic fluid. The purified material was found to contain protein, carbohydrate, and lipid and to have a general composition similar to purified preparations of PR8 influenza virus. A typical preparation of normal material had an isoelectric point at pH 2.3. Sedimentation studies indicated that the normal material can give a variety of sedimentation constants depending upon the concentration and viscosity of the preparations. The sedimentation constant, corrected for viscosity, of the major component of a fresh preparation was 170 S. The diameters of the predominant particles shown in electron micrographs of the normal material and of preparations of PR8 influenza virus were about 40 and 100 mmicro, respectively. Serological tests indicated that the normal material is a good antigen and that preparations of both A and B types of influenza virus obtained from allantoic fluids by centrifugation show a strong serological relationship to the normal material. Freezing and thawing of allantoic fluid, and repeated adsorption of virus on red cells, failed to provide a practical basis for the separation of normal protein from the virus entity in the case of PR8 virus. In the cases of similar preparations of F12 and of Lee viruses, a partial separation of a small component was accomplished by fractional centrifugation and this component and the normal protein were shown to be identical or very closely related. Antiserum to the purified normal material inhibited red cell agglutination by A and B types of influenza virus at serum dilutions of 600 to 700, but failed to show significant neutralizing capacity in chick embryo and in mouse tests at a serum dilution of 100. Rabbit antiserum to purified preparations of PR8 virus gave a 50 per cent red cell agglutination inhibition endpoint at a serum dilution of 112,000. Some of the implications of the findings are discussed.

AN EVALUATION OF METHODS FOR THE CONCENTRATION AND PURIFICATION OF INFLUENZA VIRUS

The concentration and purification of influenza virus by means of differential centrifugation in a vacuum type centrifuge, by adsorption on and elution from adult chicken red cells, by elution of the precipitate formed on freezing and thawing of allantoic fluid, by adsorption on and elution from embryonic chick red cells, and by combinations of the first method with each of the three succeeding methods, have been studied. Over-all yields of virus of about 50 to 70 per cent were obtained by these methods and combinations of methods except for somewhat lower yields when adsorption on and elution from adult chicken red cells was employed. However, the purified products obtained by methods involving only the use of red cells or the freezing and thawing technique were found to contain about 80 per cent of non-virus protein. The purified products obtained when differential centrifugation was used either alone or in combination with any one of the other methods were found to be indistinguishable and to consist of a fairly homogeneous component having a sedimentation constant of about 600 S. Such preparations possessed about 22,000 chicken red cell agglutinating units per mg. of protein nitrogen and solutions containing only about 10(-14) gm. of the materials gave 50 per cent infectivity end points in chick embryos. The Sharples centrifuge was found to be almost as efficient as the vacuum type centrifuge for the concentration and purification of influenza virus and, because of its larger capacity, is recommended for the preparation of purified virus on a large scale.

A Simplified Procedure for the Concentration and Purification of Influenza Virus

Objectives Accurate measurement of syndesmophyte development and growth in ankylosing spondylitis (AS) is needed for studies of biomarkers and of treatments to slow spinal fusion. We tested the longitudinal validity and sensitivity to change of quantitative measurement of syndesmophytes using CT. Methods We performed lumbar spine CT scans on 33 patients with AS at baseline, 1 year and 2 years. Volumes and heights of syndesmophytes were computed in four intervertebral disk spaces. We compared the computed changes to a physician9s ratings of change based on CT scan inspection. Sensitivity to change of the computed measures was compared with that of the modified Stoke AS Spinal Score (radiography) and a scoring method based on MRI. Results At years 1 and 2, respectively 24 (73%) and 26 (79%) patients had syndesmophyte volume increases by CT. At years 1 and 2, the mean (SD) computed volume increases per patient were, respectively 87 (186) and 201 (366) mm3. Computed volume changes were strongly associated with the physician9s visual ratings of change (p Conclusions CT-based syndesmophytes measurements had very good longitudinal validity and better sensitivity to change than radiography or MRI. This method shows promise for longitudinal clinical studies of syndesmophyte development and growth.