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Lecture 12 viral vaccines-1
- 2. Examples of virus vaccines produced in large quantities Human Veterinary Polio Foot-and-mouth disease Measles Marek's disease Mumps Newcastle disease Rubella Rinderpest Yellow fever Rabies Rabies Canine distemper Influenza Swine fever Blue tongue Fowl pox
- 3. Protein capsid Nucleic acid Capsomere An icosohedron virus particle Fig. 12.1
- 4. Lytic cycle of viral infection Fig. 12.2
- 5. Phases of viral growth in cell culture Phase 1 = adsorption/ penetration Phase 2 = synthesis Phase 3 = assembly Phase 4 = release Fig. 12.3 10 7 10 6 10 5 10 4 10 3 Virus titre (pfu/ ml) 2 4 6 8 10 12 Time after infection (h) 0
- 6. Reovirus (type 1) propagation on Vero cells on microcarriers
- 7. Reovirus (type 3) propagation on Vero cells on microcarriers
- 8. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines
- 9. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines
- 12. Small pox – the first weapon of mass destruction?
- 14. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> rabies
- 19. Technician at Cutter Laboratories Inspecting Filters during the Manufacture of Polio Vaccine, 1955.
- 23. Vero cells 100x magnification TEM micrograph of poliovirus
- 38. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p134
- 40. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p125
- 41. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p127
- 42. 1. Cell culture 2. Primary separation 3. Initial enrichment 4. Main purification 5. Final purification 6. Formulation 7. Final dose form - Centrifugation, microfiltration - Ultrafiltration, salt preciptitation - Various chromatography techniques - Gel filtration -> final refining steps -> removes cells -> removes water and salts -> removes majority of contaminants -> removes aggregateHis, remaining impurities - Sterile filtration, lyophilization
Hinweis der Redaktion
- Edward Jenner was an English country doctor who pioneered vaccination, Discovered in 1796 that inoculation with cowpox conferred immunity to small pox. Jenner removed fluid from the sores of a cow pox-infected milk maid. He then inoculated a farmers son with the fluid. He cut the boys arm and poured some of the fluid in. The boy contracted cow pox, but was okay. 6 weeks later, Jenner inoculated the boy with small pox virus, the boy survived (unveil painting under cows).
- The final and most famous success of Pasteur’s research was the development of the vaccine against rabies. Initially, dogs were injected with the saliva from infected animals, variable and unpredictable. Later recognized that infectious agent was in the spinal cord and brain (looked under a microscope). Injection of fluid into dogs brains produced rabies. Test animals injected with suspensions of spinal cord of rabid rabbits (attenuated by drying – the longer the drying time, the less potent in production of rabies). Dogs injected with increasingly potent preparations of minced spinal cord over time (12 days), eventually immune to full blown rabies infection. First patient was Joseph Meister in 1886, who was badly mauled by a rabid dog, couldn’t even walk – first successful vaccination Joseph Meister worked as an employee at the Pasteur Institute as a gatekeeper. In 1940, the Germans who were occupying Paris ordered him to open up the door to Pasteur’s crypt. Rather than comply, he committed suicide.
- Death of rabies-infected humans and animals is a result of alterations to the neurons. Rabies RNA competes with host RNA, impairing neural functions. Bodies’ response to rabies, production of nitric oxide, may act as a toxin to the central nervous system. Determining factor of rabies is the glycoprotein that makes up the viral membrane. Amino acid at position 333 is critical (arginine or lysine): substitution at this site attenuates the virus, spreads more slowly in the central nervous system and is not able to infect certain types of nerve cells.
- Inactivated vaccine – licensed in 1955, used until the early 1960’s Grown in monkey kidney tissue (Vero cell line), inactivated with formaldehyde Supplied in a single syringe dose (subcutaneous or intromuscular) Oral vaccine Live attenuated strains, grown in Vero cells No shots required Intestinal and bodily immunity (sense ingested); Salk’s only bodily Salk’s – person still a carrier Sabin – lifelong immunity, no booster required
- The virus is inactivated by heat or by chemical treatment, using formaldehyde. Have to be careful, excessive treatment will remove immunogenicity (can’t get immune response), while undertreatment may result in infectious virus that can still cause disease. This happened with Salk’s polio vaccine. For inactivated viruses, you require multiple doses. Prepared from attenuated strains that are almost or completely non-pathogenic, but are still capable of inducing an immune response. They multiply in the human host and provide continuous antigenic stimulation over time. You don’t need as many doses as you do with killed viral vaccines, and is very good in provoking a strong immune response. Highly purified vaccines containing the antigenic determinants from viruses may be used. This is a method used to produce vaccines for influenza A and B (the flu).
- Synthetic man made proteins using known amino sequences, if you know the immunogenic sites on a virus. Not applicable for all viruses, can’t be used to produces polio vaccines, since the antigenic sites were made up of two or more viral capsid proteins forming a specific 3-D conformation.
- There are many risks associated with the use of animal cells for the production of biological products. Animal cells share many biochemical and physiological similarities to cells found in human tissues. Animal cells are vulnerable to infections by viruses as well as to events that may cause oncogenesis, leading to tumor formation and cancer. Because of these risks, initially only normal primary cells were used to produce biological products, such as viral vaccines. These were not optimal for industrial conditions as you needed to produce fresh cell stocks from animal material for each production run – get batch to batch variability, slower and more expensive. Years later, it was accepted grudgingly that transformed cells, continuous cell lines, would have to be used (infinite growth, less dependence on complex medium, grow in suspension). These types of cells can be cancerous if transferred to animal tissue, but so long as the protein or antibody product was purified and well characterized, the biological risks can be eliminated.
- Protein may be derived from the production cells that have leaked into the media, or from components from the media (serum) Protein contamination may lead to an allergic response or a transformation event. Test for impurities using antibodies against known proteins (i.e. proteins found in serum) Perform a mock purification by performing purification procedures on the growth medium to look for proteins that may be derived from the production cells. The cells in this case would not have the gene encoding the protein of interest. You are just looking for any residual proteins produced by the cells that you normally may not see when you have the protein of interest being produced.
- In the mid-80’s, it was decided that 10 pg of exogenous DNA per dose of drug was acceptable, since you require about 100 pg of oncogene to trigger a transformation event. To insure that DNA is being removed, radioactive DNA is introduced into the medium, and it’s removal at each purification step is monitored, to show that DNA levels can be reduced to acceptable limits. -hybridization of DNA: bound to membrane and hybridized to species specific probe, compared to DNA-hybrid standards
- Endogenous viruses are those that have integrated into the genome (prophage). Cell characterization includes karyotyping, isoenzyme analysis, and immunological analysis, DNA finger-printing Must make sure that cell characteristics don’t change over the production run, compared to profile of the cells are identical to the cells stored in the Master cell bank. Alterations may indicate cross-contamination of the cells or changes to the cell line during the production process .
- Must be aware of possible changes to the recombinant protein, including variations to the amino acid sequence, glycosylation of the protein as well as changes to the amino acids. These properties are compared to the naturally occurring protein. Post translational modifications can change the biopharmaceutical properties of the protein Also look at changes that may occur during production of the protein in the medium – oxidation of aa residues, breaking and reforming of sulfide bonds, blocking of N- and C-terminals.
- Various tests are conducted to insure that the protein meets expectations for pharmaceutical activity. Tests are also conducted to characterize the protein and to check to make sure the protein is pure. Various methods can also be used to check and see if the protein is correctly folded, and that the proper glycans are attached to the protein. The protein must also be tested for contaminants (as we’ve discussed) such as protein, DNA, viruses, etc.