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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

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

Protein capsid

Nucleic acid

Capsomere

An icosohedron virus particle

Fig. 12.1

Lytic cycle of viral infectionFig. 12.2

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

Reovirus (type 1) propagation on Vero cells on microcarriers



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


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

Small pox – the first weapon of mass destruction?


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


Animal cell products: Viral vaccines → rabies


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


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

Technician at Cutter Laboratories Inspecting Filters during the Manufactureof Polio Vaccine, 1955.

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)

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

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

Vero cells 100x magnification

TEM micrograph of poliovirus

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

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

Wild Poliovirus 2004


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


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)



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)


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



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


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


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



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



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)



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



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

Cartwright, T. 1994. Animal cells as bioreactors. Cambridge:Cambridge University Press. p134


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

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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