ANIMAL CELL CULTURE

LESSON 21 CELL CULTURE BASED VACCINES

Hi!!after a long discussion relating to the bulk production and storage of animal cell cultures,it is now time to talk about the applications of animal cell cultures. Scientists studying the biochemistry and biophysics of cell growth and division have made extensive use of cultured animal cells, as have those engaged in research on animal viruses. However, the value of cultured animal cells goes far beyond this, for they produce a wide range of biological products of com-mercial interest including immunoregulators, anti-bodies, polypeptide growth factors, enzymes and hormones. Already they are used in the manufacture of virus vaccines, tissue plasminogen activator (an enzyme which facilitates the destruction of blood clots), interferon-a (for the treatment of cancer), monoclonal antibodies and tumourspecific antigens (for inclusion in diagnostic kits). Consideration is also being given to the possibility of growing fibroblasts in culture from, for example, burns patients. These cultured cells would be used as a reconstituent skin to facilitate wound healing. Another potential application of cul-tured cells is their use in evaluating new drugs and toxic chemicals. When used in concert with conven-tional animal models they could reduce much of the time, cost and effort as well as the total number of animals. Even the cells themselves can have com-mercial value. For example, cultured fibroblasts from burns patients have been used as a reconstituted skin to facilitate wound healing. Production of Viral Vaccines: The oldest commercial application of animal cell cultures is for the production of viral vaccines and it could be argued that globally, with the possible exception of antibiotics, they have provided greater benefit than any other pharmaceutical product. The deployment of a single vaccine, that against smallpox, has been of immense benefit to mankind and hopefully has eliminated the virus for evermore. Because viruses are obligate intracellular parasites the earliest viral vaccines, those against smallpox and rabies, were made in intact animals such as calves, sheep and rabbits. Today the only intact host used in advanced production techniques is the developing chick embryo for the manufacture of influenza and yellow fever vaccines. All other vaccines are prepared by the growth of virus in cell culture. Table 1 lists the major human and veterinary virus vaccines and the cell cultures used in their production. Viral vaccines are of two types. Those which are prepared from live, avirulent (attenuated) organisms often give lifelong protection but carry with them the attendant risk of contamination with other viruses derived from the cell culture. Clearly, rigorous micro-biological and other quality control procedures are essential. Vaccines prepared from killed (inactivated) viruses are less satisfactory since they are not as immunogenic as live vaccines, presumably because the virus does not multiply in the animal host, and have to be injected repeatedly according to carefully balanced immunization schedules. The only inactivated vaccines for human use are those against rabies and, in some
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instances, polio. The usual inactivating agents are dilute formalin or |3-propiolactone. In theory, the production of viral vaccines is rela-tively simple. The cell cultures principally used for virus vaccine production are prepared from monkey kidney or chick embryos. More recently there has been an increasing use of human diploid cells. Table 1 Some viral vaccines currently available for human and veterinary use.
Human vaccines Virus Measles Cell used for culture Veterinary vaccines Virus

Polio (inactivated) Polio (live) Rabies Rubella

Cell used for culture Chick embryo Canine Chick fibroblasts distemper embryo Fibroblasts or dog Monkey kidney Canine hepatitis Dog cells kidney Monkey kidney or Foot and Bovine human diploid mouth kidney cells disease cells Human diploid Rabies Duck cells embryo or Chick Rabbit kidney, Feline Cat kidney duck panleucopenia cells embryo or human Marek's disease Chick diploid cells embryo cells

Since these cells grow as monolayers all that is required in vaccine preparation is to infect them with the appropriate virus and then harvest the culture fluid after virus multiplication has occurred. The culture fluid contain-ing the virus is clarified by filtration. In the case of killed-virus vaccines a concentration step is usually employed after the inactivation step. When processing is complete the virus suspension is blended with stabilizers to prevent loss of potency, a problem with live attenuated vaccines, and stored at low temperature. A typical production process is shown in Fig

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ANIMAL CELL CULTURE

Fig:Production of killed-Virus Vaccines

Constraints in the preparation of viral vaccines :

In practice, the production of a virus vaccine is far more complex, not just because of the labour involved in large-scale animal culture but because of the rigor-ous quality control and safety procedures which are required; for example, all tissue culture substrates are rigorously examined to exclude contamination with infectious agents from the source animal or, in the case of human diploid cells, to exclude abnormal cellular characteristics. Monkey tissues are tested exhaustively for extraneous agents, e.g. herpes virus B, simian virus 40, tubercle bacilli and mycoplasmas. Human diploid cells are subjected to detailed karyological examination to exclude cultures with features resembling trans-formed cell lines or malignant tissues. Safety of the final product is another important consideration. With killed-virus vaccines the potential hazards are those due to incomplete virus inactivation - a possible cause of recent outbreaks of foot and mouth disease in Europe! The tests used to detect live virus consist of the inoculation of susceptible tissue culture and of susceptible animals. The cultures are examined for cytopathic effects and animals for symptoms of disease and histological evidence of infection at autopsy. With attenuated viral vaccines the potential hazards are those associated with reversion of the virus to a degree of virulence capable of causing disease in vaccinees. This possibility is minimized by the use of stable virus seed stocks but virulence checks on the final product are necessary; for example, in the production of attenuated poliomyelitis vaccine the neurovirulence of each batch following intraspinal inoculation of monkeys is compared with that of a control vaccine. Many of the problems associated with the production of viral vaccines, particularly the safety aspects, may disappear now that recombinant DNA technology can be used to enable virus subunits to be produced in bacteria or yeasts. Another advantage of recombinant DNA technology is that it may permit the
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production of vaccines against those viruses which cannot be grown in cell culture to a litre high enough to provide suf-ficient antigen for effective vaccination. Notes:

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