Lecture 12 viral vaccines-1

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Industrial Microbiology Dr. Butler 2011

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  • 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.
  • Lecture 12 viral vaccines-1

    1. 1. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines <ul><li>virus – a genetic element that contains RNA or DNA which replicates in cells (hosts) but is characterized by an extracellular state (particle consisting of genetic material surrounded by a protein coat and possibly other macromolecular components </li></ul><ul><li>cell cultures are convenient for viral research because cell material is continuously available for research </li></ul><ul><li>organ cultures may also be used as they permit growth of viruses under controlled laboratory conditions </li></ul>
    2. 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. 3. Protein capsid Nucleic acid Capsomere An icosohedron virus particle Fig. 12.1
    4. 4. Lytic cycle of viral infection Fig. 12.2
    5. 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. 6. Reovirus (type 1) propagation on Vero cells on microcarriers
    7. 7. Reovirus (type 3) propagation on Vero cells on microcarriers
    8. 8. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines
    9. 9. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines
    10. 10. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> smallpox <ul><li>1796 – a vaccine (cowpox) to smallpox was described by Edward Jenner </li></ul><ul><li>-> Jenner infected a young boy with cowpox; six weeks later Jenner infected the boy with smallpox </li></ul><ul><li>-> the term vaccine, from the Latin vacca for cow </li></ul>
    11. 11. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> smallpox <ul><li>small pox was a serious, contagious, and sometimes fatal infectious disease </li></ul><ul><li>Symptoms include bumps on face and body of an infected person, rash, high fever </li></ul><ul><li>-> incubation period varies from 2-17 days </li></ul><ul><li>spread by direct and prolonged face-to-face contact, infected bodily fluids or contaminated objects such as bedding or clothing </li></ul><ul><li>last case of small pox occurred in Somalia in 1977, disease eliminated </li></ul>
    12. 12. Small pox – the first weapon of mass destruction?
    13. 13. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> rabies <ul><li>1885 – Louis Pasteur developed a vaccine to the rabies virus, which infects humans and animals </li></ul><ul><li>extracts from the spinal cord of rabid dogs were applied to the brains of test dogs, induced rabies </li></ul><ul><li>suspensions of the spinal cord of rabid rabbits were injected into test animals; solution was attenuated by air drying in a Roux bottle for 12 days </li></ul>
    14. 14. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> rabies
    15. 15. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> polio <ul><li>1949 – John Enders and his colleagues discovered the poliomyelitis virus could be grown from human embryonic cells, awarded the Nobel Prize in 1954 </li></ul><ul><li>-> virus extracted from mouse brain tissues and injected into mice and monkeys, inducing paralysis typical of polio </li></ul><ul><li>1954 – first human vaccine (polio) produced using large scale animal cell cultures (primary monkey kidney cells) </li></ul><ul><li>-> one of the first commercial products of cultured animal cells </li></ul>
    16. 18. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> polio <ul><li>Jonas Salk developed a vaccine by inactivating 2/3 strains of polioviruses, using formalin </li></ul><ul><li>-> children still developed polio after injections of vaccine developed by Salk and the Cutter Company </li></ul><ul><li>-> vaccine was not properly inactivated </li></ul><ul><li>Albert Sabin developed an attenuated polio vaccine that could be administered orally rather than injected </li></ul><ul><li>-> vaccine placed on sugar cubes or teaspoon of syrup </li></ul>picornavirus
    17. 19. Technician at Cutter Laboratories Inspecting Filters during the Manufacture of Polio Vaccine, 1955.
    18. 20. Poliovirus Vaccine <ul><li>1955 Inactivated vaccine </li></ul><ul><li>1961 Types 1 and 2 monovalent OPV </li></ul><ul><li>1962 Type 3 monovalent OPV </li></ul><ul><li>1963 Trivalent OPV </li></ul><ul><li>1987 Enhanced-potency IPV (IPV) </li></ul>
    19. 21. Inactivated Polio Vaccine <ul><li>Contains 3 serotypes of vaccine virus </li></ul><ul><li>Grown on monkey kidney (Vero) cells </li></ul><ul><li>Inactivated with formaldehyde </li></ul><ul><li>Contains 2-phenoxyethanol, neomycin, streptomycin, polymyxin B </li></ul>
    20. 22. Oral Polio Vaccine <ul><li>Contains 3 serotypes of vaccine virus </li></ul><ul><li>Grown on monkey kidney (Vero) cells </li></ul><ul><li>Contains neomycin and streptomycin </li></ul><ul><li>Shed in stool for up to 6 weeks following vaccination </li></ul>
    21. 23. Vero cells 100x magnification TEM micrograph of poliovirus
    22. 24. <ul><li>Poliomyelitis—United States, 1950-2005 </li></ul>Inactivated vaccine Live oral vaccine Last indigenous case
    23. 25. Polio Eradication <ul><li>Last case in United States in 1979 </li></ul><ul><li>Western Hemisphere certified polio free in 1994 </li></ul><ul><li>Last isolate of type 2 poliovirus in India in October 1999 </li></ul><ul><li>Global eradication goal </li></ul>
    24. 26. Wild Poliovirus 2004
    25. 27. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> foot and mouth disease <ul><li>highly infectious viral disease of </li></ul><ul><li>cloven hoofed animals </li></ul><ul><li>major economic consequences </li></ul><ul><li>humans are carriers, transmit to </li></ul><ul><li>healthy animals </li></ul>
    26. 28. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines -> foot and mouth disease <ul><li>seven different types of foot and mouth disease, 60 subtypes </li></ul><ul><li>-> no universal vaccine </li></ul><ul><li>symptoms include salivation, depression, anorexia, loss of appetite lameness, and the presence of blisters in mouth and body, inflamed tissues under the hooves (hooves may be shed) </li></ul><ul><li>-> incubation period lasts 2-21 days </li></ul><ul><li>spread by movement of infected animals, infected feed, vehicles and facilities, infected water, inhalation, infected humans (carriers) </li></ul>
    27. 29. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines <ul><li>Vaccinations – injection of viral antigen in non-pathogenic form to induce antibody response </li></ul><ul><li>-> antibodies can then protect against live pathogenic form of virus </li></ul>An electron micrograph of a rotavirus particle (A) and a rotavirus reacted with antibody (B)
    28. 30. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines <ul><li>1. inactivated pathogenic virus – chemically or heat inactivated </li></ul><ul><li>2. attenuated live virus – non-pathogenic, surface still contains proteins that can elicit an immune response (i.e. viral capsid protein) </li></ul><ul><li>-> attenuated viruses can become virulent </li></ul><ul><li>3. peptides which mimic antigenic effects of surface protein </li></ul><ul><li>-> higher quantities required to invoke response </li></ul><ul><li>-> useful as some viruses cannot be cultured </li></ul>
    29. 31. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines <ul><li>4. synthetic peptides </li></ul><ul><li>-> precisely defined and free from unnecessary components associated with side effects (nucleic acids, viral or external proteins) </li></ul><ul><li>-> not applicable for all viruses </li></ul><ul><li>-> less immunogenic, may require adjuvants, boosters </li></ul><ul><li>5. DNA vaccines – injection of DNA encoding viral proteins directly into animal </li></ul><ul><li>-> inexpensive, easy to produce </li></ul><ul><li>-> in theory extremely safe, free of side effects </li></ul><ul><li>-> clinical trials involving HIV, influenza, herpes simplex virus </li></ul>
    30. 32. Lecture 13 Animal Cell Biotechnology Animal cell products: Viral vaccines <ul><li>Safety concerns </li></ul><ul><li>tumorigenic cell lines (i.e. HeLa) considered a no-no </li></ul><ul><li>primary monkey kidney cells initially used to produce polio vaccine </li></ul><ul><li>-> contaminated with tumorigenic virus SV40 </li></ul><ul><li>1960’s normal human diploid fibroblasts used to produce vaccines </li></ul><ul><li>-> human lung fibroblast lines (WI-38 and MRC-5) used for polio vaccine </li></ul><ul><li>Vero (African green monkey cells) first continuous line to produce human vaccine products, including polio vaccine </li></ul>
    31. 33. Lecture 13 Animal Cell Biotechnology Animal cell products : Safety precautions <ul><li>Potential risks of products derived from animal cell cultures: </li></ul><ul><li>1. process derived proteins </li></ul><ul><li>2. residual DNA contamination </li></ul><ul><li>3. viral contamination </li></ul><ul><li>4. safety of the cell line </li></ul>
    32. 34. Lecture 13 Animal Cell Biotechnology Animal cell products : Safety precautions <ul><li>1. process derived proteins </li></ul><ul><li>residual proteins derived from the production cells could trigger an immune response or a transformation event </li></ul><ul><li>may stimulate host allergic responses </li></ul><ul><li>screen for protein impurities using antibodies </li></ul><ul><li>perform “mock” purification -> check for impurities in supernatant derived from non-producing cells </li></ul>
    33. 35. Lecture 13 Animal Cell Biotechnology Animal cell products : Safety precautions <ul><li>2. residual DNA contamination </li></ul><ul><li>potential for DNA carrying oncogenes, which could lead to: </li></ul><ul><li>-> tumorigenesis </li></ul><ul><li>-> uptake and expression of viral genes </li></ul><ul><li>-> insertion of exogenous sequences into critical control regions of the genome, altering expression of certain genes </li></ul><ul><li>residual DNA should be reduced to a minimal and safe level (< 10 pg/dose of injectable product) </li></ul>
    34. 36. Lecture 13 Animal Cell Biotechnology Animal cell products : Safety precautions <ul><li>3. viral contamination </li></ul><ul><li>endogenous viruses, esp. retroviruses, are a potential hazard </li></ul><ul><li>chemical and physical treatments used to inactivate contaminating viruses </li></ul><ul><li>may test purification process by adding known viruses and following loss of viability </li></ul>
    35. 37. Lecture 13 Animal Cell Biotechnology Animal cell products : Safety precautions <ul><li>4. safety of the cell line </li></ul><ul><li>continuous (immortal) cell lines often used to produce recombinant protein products </li></ul><ul><li>cells have activated oncogenes and many harbor endogenous viruses </li></ul><ul><li>extensive cellular characterization and the ability to detect low amounts of known contaminants (i.e. DNA) have shifted focus to ensuring final products not contaminated and risk free </li></ul>
    36. 38. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p134
    37. 39. Lecture 13 Animal Cell Biotechnology Animal cell products: Safety precautions <ul><li>Characterization of recombinant protein products </li></ul><ul><li>recombinant proteins and monoclonal antibodies must satisfy the same quality, safety, and efficacy criteria as other pharmaceutical products </li></ul><ul><li>must be well characterized, consistently produced from batch to batch </li></ul><ul><li>any possible contaminant must be identified and consistent </li></ul>
    38. 40. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p125
    39. 41. Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p127
    40. 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

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