lecture 12 viral vaccines-1
DESCRIPTION
Industrial Microbiology Dr. Butler 2011TRANSCRIPT
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines
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
• cell cultures are convenient for viral research because cell material is continuously available for research
• organ cultures may also be used as they permit growth of viruses under controlled laboratory conditions
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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
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Protein capsid
Nucleic acid
Capsomere
An icosohedron virus particle
Fig. 12.1
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Lytic cycle of viral infectionFig. 12.2
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Phases of viral growth in cell culture
107
106
105
104
103
Vir
us ti
tre
(pfu
/ ml)
2 4 6 8 10 12
Time after infection (h)
0Phase 1 = adsorption/ penetrationPhase 2 = synthesisPhase 3 = assemblyPhase 4 = release
Fig. 12.3
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Reovirus (type 1) propagation on Vero cells on microcarriers
Reovirus (type 1) propagation on Vero cells on microcarriers
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Reovirus (type 3) propagation on Vero cells on microcarriers
Reovirus (type 3) propagation on Vero cells on microcarriers
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → smallpox
• 1796 – a vaccine (cowpox) to smallpox was described by Edward Jenner
→ Jenner infected a young boy with cowpox; six weeks later Jenner infected the boy with smallpox
→ the term vaccine, from the Latin vacca for cow
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → smallpox
• small pox was a serious, contagious, and sometimes fatal infectious disease
• Symptoms include bumps on face and body of an infected person, rash, high fever
→ incubation period varies from 2-17 days
• spread by direct and prolonged face-to-face contact, infected bodily fluids or contaminated objects such as bedding or clothing
• last case of small pox occurred in Somalia in 1977, disease eliminated
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Small pox – the first weapon of mass destruction?
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → rabies
• 1885 – Louis Pasteur developed a vaccine to the rabies virus, which infects humans and animals
• extracts from the spinal cord of rabid dogs were applied to the brains of test dogs, induced rabies
• 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
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → rabies
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → polio
• 1949 – John Enders and his colleagues discovered the poliomyelitis virus could be grown from human embryonic cells, awarded the Nobel Prize in 1954
→ virus extracted from mouse brain tissues and injected into mice and monkeys, inducing paralysis typical of polio
• 1954 – first human vaccine (polio) produced using large scale animal cell cultures (primary monkey kidney cells)
→ one of the first commercial products of cultured animal cells
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → polio
• Jonas Salk developed a vaccine by inactivating 2/3 strains of polioviruses, using formalin
→ children still developed polio after injections of vaccine developed by Salk and the Cutter Company
→ vaccine was not properly inactivated
• Albert Sabin developed an attenuated polio vaccine that could be administered orally rather than injected
→ vaccine placed on sugar cubes or teaspoon of syrup
picornavirus
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Technician at Cutter Laboratories Inspecting Filters during the Manufactureof Polio Vaccine, 1955.
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Poliovirus Vaccine• 1955 Inactivated vaccine
• 1961 Types 1 and 2 monovalent OPV
• 1962 Type 3 monovalent OPV
• 1963 Trivalent OPV
• 1987 Enhanced-potency IPV (IPV)
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Inactivated Polio Vaccine• Contains 3 serotypes of vaccine virus• Grown on monkey kidney (Vero) cells• Inactivated with formaldehyde• Contains 2-phenoxyethanol, neomycin,
streptomycin, polymyxin B
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Oral Polio Vaccine• Contains 3 serotypes of vaccine virus• Grown on monkey kidney (Vero) cells• Contains neomycin and streptomycin• Shed in stool for up to 6 weeks following
vaccination
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Vero cells 100x magnification
TEM micrograph of poliovirus
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0
5000
10000
15000
20000
25000
1950 1956 1962 1968 1974 1980 1986 1992 1998 2004
Cases
Poliomyelitis—United States, 1950-2005
Inactivated vaccine
Live oral vaccine
Last indigenous case
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Polio Eradication• Last case in United States in 1979• Western Hemisphere certified polio free in
1994• Last isolate of type 2 poliovirus in India in
October 1999• Global eradication goal
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Wild Poliovirus 2004
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → foot and mouth disease
• highly infectious viral disease of
cloven hoofed animals
• major economic consequences
• humans are carriers, transmit to healthy animals
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines → foot and mouth disease
• seven different types of foot and mouth disease, 60 subtypes
→ no universal vaccine• 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)
→ incubation period lasts 2-21 days• spread by movement of infected animals, infected feed,
vehicles and facilities, infected water, inhalation, infected humans (carriers)
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines
Vaccinations – injection of viral antigen in non-pathogenic form to induce antibody response
→ antibodies can then protect against live pathogenic form of virus
An electron micrograph of a rotavirus particle (A) and a rotavirus reacted with antibody (B)
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines1. inactivated pathogenic virus – chemically or heat
inactivated
2. attenuated live virus – non-pathogenic, surface still contains proteins that can elicit an immune response (i.e. viral capsid protein)
→ attenuated viruses can become virulent
3. peptides which mimic antigenic effects of surface protein
→ higher quantities required to invoke response
→ useful as some viruses cannot be cultured
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccines
4. synthetic peptides → precisely defined and free from unnecessary
components associated with side effects (nucleic acids, viral or external proteins)
→ not applicable for all viruses → less immunogenic, may require adjuvants, boosters
5. DNA vaccines – injection of DNA encoding viral proteins directly into animal
→ inexpensive, easy to produce → in theory extremely safe, free of side effects → clinical trials involving HIV, influenza, herpes simplex
virus
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Viral vaccinesSafety concerns• tumorigenic cell lines (i.e. HeLa) considered a no-no
• primary monkey kidney cells initially used to produce polio vaccine
→ contaminated with tumorigenic virus SV40
• 1960’s normal human diploid fibroblasts used to produce vaccines
→ human lung fibroblast lines (WI-38 and MRC-5) used for polio vaccine
• Vero (African green monkey cells) first continuous line to produce human vaccine products, including polio vaccine
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Lecture 13 Animal Cell Biotechnology
Animal cell products : Safety precautions
Potential risks of products derived from animal cell cultures:
1. process derived proteins
2. residual DNA contamination
3. viral contamination
4. safety of the cell line
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Lecture 13 Animal Cell Biotechnology
Animal cell products : Safety precautions
1. process derived proteins
• residual proteins derived from the production cells could trigger an immune response or a transformation event
• may stimulate host allergic responses
• screen for protein impurities using antibodies
• perform “mock” purification → check for impurities in supernatant derived from non-producing cells
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Lecture 13 Animal Cell Biotechnology
Animal cell products : Safety precautions
2. residual DNA contamination
• potential for DNA carrying oncogenes, which could lead to:
→ tumorigenesis
→ uptake and expression of viral genes
→ insertion of exogenous sequences into critical control regions of the genome, altering expression of certain genes
• residual DNA should be reduced to a minimal and safe level (< 10 pg/dose of injectable product)
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Lecture 13 Animal Cell Biotechnology
Animal cell products : Safety precautions
3. viral contamination
• endogenous viruses, esp. retroviruses, are a potential hazard
• chemical and physical treatments used to inactivate contaminating viruses
• may test purification process by adding known viruses and following loss of viability
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Lecture 13 Animal Cell Biotechnology
Animal cell products : Safety precautions
4. safety of the cell line
• continuous (immortal) cell lines often used to produce recombinant protein products
• cells have activated oncogenes and many harbor endogenous viruses
• 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
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Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p134
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Lecture 13 Animal Cell Biotechnology
Animal cell products: Safety precautions
Characterization of recombinant protein products
• recombinant proteins and monoclonal antibodies must satisfy the same quality, safety, and efficacy criteria as other pharmaceutical products
• must be well characterized, consistently produced from batch to batch
• any possible contaminant must be identified and consistent
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Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p125
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Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p127
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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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