We have studied the influence of glycosaminoglycans on cell adsorption of, and plaque formation by, HSV-1 and HSV-2, with regard to the role of saccharide structure, chain length and charge density. Heparin and highly-sulphated heparan sulphate (1.5 sulphate groups/disaccharide unit), but neither chondroitin sulphate nor dermatan sulphate, were found to compete with the cellular receptor for attachment of HSV.
Rabbit pox virus had the lowest iso-electric point at approx. 2″3. Variola, alastrim and monkey pox viruses all had iso-electric points of approx. 3″4 but at greater pH than this monkey pox showed somewhat higher mobility than the other two viruses.
For efficient production methods, particularly in sight of constantly increasing economic pressure within human healthcare systems, highly productive downstream schemes have to be developed. Here, we discuss unit operations and downstream trains to purify virus particles for use as vaccines and vectors for gene therapy.
The process involves a number of complex tasks and unit operations ranging from selection of host cells and virus strains for the cultivation in bioreactors to the purification and formulation of the final product. For the majority of cell culture-derived products, efforts focused on maximization of bioreactor yields, whereas design and optimization of downstream processes were often neglected. Owing to this biased focus, downstream procedures today often constitute a bottleneck in various manufacturing processes and account for the majority of the overall production costs.
Enzymes and bioenergy production from waste substrates
Even when the mice were inoculated with MVA intracranially, they were not affected. Significant protection against a lethal dose of an orthopoxvirus was obtained in mice following immunization with the Lister strain, while larger doses and repeated vaccination procedure, were required with MVA. The Lister virus stock applied in Israel, was found to be heterogeneous in its plaque morphology. Two variants isolated from it, showed significant attenuation for mice, when inoculated intranasally and intracranially, as compared to a third variant and to the unpurified stock of the virus. The complete genomic DNA sequence of the highly attenuated vaccinia strain modified vaccinia Ankara (MVA) was determined.
The virus failed to reach the brain of the mice when inoculated intranasally at a dose of 500,000 plaque forming units, but was lethal for 50% of them, when injected intracranially. Lower doses of virus injected intracranially caused some weight loss initially, but later the mice completely recovered. Modified vaccinia virus Ankara (MVA), when infected intranasally, did not spread beyond the lungs to other organs of the mice.
The smallpox vaccination strain MVA: Marker, genetic structure, experience gained with the parentera…
This dependency may be the mechanism behind our observation that a novel viral genotype (MVA-CR) accumulates with serial passage in suspension cultures. Sequencing of a major part of the genomic DNA of the new strain revealed point mutations in three genes. We hypothesize that these changes confer an advantage because they may allow a greater fraction of MVA-CR viruses to escape the host cells for infection of distant targets. Production and purification of MVA-based vaccines may be simplified by this combination of designed avian cell line, chemically defined media and the novel virus strain. A purification scheme for cell culture-derived smallpox vaccines based on an orthogonal downstream process of pseudo-affinity membrane adsorbers (MA) and hydrophobic interaction chromatography (HIC) was investigated.
Elution from Sartobind D resulted in yields of only 38%. Lowering the pH (down to 4.2) or combining a low pH with salt displacement failed to elute virions.
Virus from diluted salt-released harvests (0.14 M NaCl) was not appreciably adsorbed onto either heparin-Sepharose or Cellufine-heparin matrices but was virtually completely adsorbed onto Cellufine-sulfate and heparin-HP matrices. Virus was recovered by either a linear salt gradient elution (0.14-2 M NaCl) or by a single-step elution with 1.5 M NaCl in phosphate buffer. Recoveries of infectious virus with step elution were 21% and 89%, respectively, for these matrices.