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Johns Hopkins Bloomberg School of Public HealthCAAT

Animals and Alternatives in Testing: History, Science, and Ethics

Joanne Zurlo, Deborah Rudacille, and Alan M. Goldberg

Appendix A: Methodologies of Vaccine Development

1796Struck by a milkmaid's observation that she would never develop smallpox as she had once had cowpox, Jenner inoculates a healthy 8-year-old boy with material from a cowpox sore on the hand of the milkmaid. When exposed to smallpox, the boy fails to develop the disease. Jenner begins a series of experiments in transferring cowpox (vaccinia virus) arm to arm. Each vaccinated individual is later proven resistant to smallpox.
1876Robert Koch obtains pure cultures of the anthrax bacillus and transmits the disease to laboratory animals; the first time a germ is definitively proven to cause a disease.
1879Louis Pasteur grows the fowl cholera bacillus in culture. Returning to his Paris laboratory after a vacation, he inoculates chickens with the aged culture and discovers that the weakened bacillus does not cause disease and that it creates immunity in the chickens to more virulent strains of cholera.
1881Pasteur applies the principles discovered in the cholera experiment to the disease anthrax, growing the anthrax bacillus in chicken broth. In May 1881, he vaccinates 25 sheep, six cows, and one goat with a series of two inoculations; the first, administered on May 5, contains a highly attenuated (weakened) form of the culture and the second, administered on May 17, a less attenuated culture. Twenty-five sheep, four cows, and one goat are used as controls and receive no vaccine. On May 31, all of the animals are given an injection of a fully virulent anthrax culture. by June 2, all of the vaccinated animals (save for one sheep) are alive and all of the controls either dead or seriously ill.
1885Unable to cultivate rabies in vitro, Pasteur produces rabies in rabbits via intracerebral injections of rabies-infected cow brain. Successive passages of the virus in rabbits reduces the incubation period from 15 days to a fixed period of seven days (after approximately 50 passages). Sacrificing the animals, Pasteur removes their spinal cords, hanging them to dry. Dogs inoculated with a series of injections of attenuated spinal cord preparations resist challenge with virulent strains of virus. On July 6, a 9-year-old boy who was bitten 14 times by a rabid dog arrives in Paris and Pasteur -- knowing that the child's death is inevitable -- attempts the procedure, which has been successful in dogs, inoculating the boy 13 times over a 10-day period with infected spinal cord preparations of increasing virulence. The boy remains healthy.
1890Behring and Kitasato observe that the serum of an animal that has received injections of living or killed broth cultures of diphtheria or tetanus bacilli soon exhibits antibodies to the toxin (antitoxin). Behring and Kitasato discover that the antibody will protect not only the directly immunized animal, but also other animals immunized with an injection of its blood serum, and further, the antibody will cure an animal in which symptoms due to the toxin have developed. The first injection into a human patient, on December 25, 1891 is successful. Guinea pigs, sheep, goats, and horses are used for the development and production of the antitoxin.
1895Kitasato and Yersin discover the bacillus that causes plague and succeed in growing the organism in culture. Yersin, Calmette, and Borrel prepare a killed vaccine by heating the bacillus at 50°c for one hour. In the next year, Haffkine prepares another plague vaccine using a broth culture of the organisms heated at 70°c and phenolized. Haffkine tests the vaccine by injecting himself and several hundred volunteers. The vaccine is successful.
1896Wilhelm Kolle introduces a heat-killed vaccine for cholera, growing the vibrios on agar, suspending them in saline and exposing them to 50°c for less than an hour. A modified version of this vaccine is still in use today.
1921Albert Calmette and Camille Guerin develop the tuberculosis vaccine BCG via prolonged cultivation of a strain of bovine (cow) tubercule bacillus on a glycerine potato medium containing ox bile. After 231 subcultures over a period of 13 years, the strain is considered attenuated and stable. The first human experiments with BCG, undertaken in July 1921, are successful.
1927Theiler and Smith isolate the highly virulent Asibi strain of yellow fever, developing the attenuated strain, 17D, via a sequence of inoculations through mice and cultivation in tissue culture.
1935Theiler and Smith introduces their yellow fever vaccine, transferring the 17D strain from chick tissue culture to the developing egg to facilitate large-scale production. During World War II large quantities of the vaccine are prepared with the 17D strain.
1937Zinsser develops a killed vaccine for typhus from rickettsiae grown in the peritoneal cavity of rats. In 1939, Zinsser, FitzPatrick, and Wei obtain cultures of rickettsiae of typhus by culturing the organism with chick or embryo mouse tissue on agar. By 1940, Harold Cox had succeeded in growing rickettsiae in the yolk sac of the developing chick embryo, preparing the way for mass production of a killed vaccine for typhus. It should also be noted that cleanliness prevents the growth and spread of typhus by destroying its messengers, lice.
1949Enders, Weller, and Robbins establish that the poliomyelitis virus can be grown in cultures of human embryonic and adult non-nervous tissues. They later discover that monkey tissues, particularly kidneys, also form a good culture medium for the development of the virus for vaccine production.
1952Jonas Salk develops a killed-virus vaccine against polio using tissue cultures of the monkey testicle or kidney.
1953Albert Sabin produces a live, attenuated polio vaccine and carries out tests on 10,000 monkeys, 160 chimpanzees, and 243 humans, including his own family.
1956Isaacs and Lindenmann discover interferon, a protein produced in animal cells to fight viruses, first observed in a chick embryo tissue culture of the influenza virus, and later isolated in a pure form.
1958Milovanovic, Enders, Mitus, and Katz succeed in passaging a strain of measles virus more than 50 times in human tissue cultures, and then adapting it to chick embryo tissue culture. The vaccine is successful but produces a mild reaction. Simultaneous injection with the vaccine and gamma globulin reduces the reaction.
1962Thomas Weller reports that he has successfully cultivated the rubella virus in tissue culture. After serial passage in tissue culture several attenuated strains become available.
1966Meyer and Parman develop a live-virus vaccine for rubella.
1981Acquired immune deficiency syndrome (AIDS) is recognized by the U.S. Centers for Disease Control.
1983Montagnier and colleagues discover what they believe to be the AIDS virus and call it lymphadenopathy-associated virus (LAV).
1984Gallo and colleagues describe the development of cell lines permanently infected with the AIDS virus, which they call human T-cell lymphotropic virus-III (HTLV III).
1986The International Committee on the Taxonomy of Viruses recommends that the retrovirus believed to cause AIDS be renamed human immunodeficiency virus (HIV).
1992The U.S. National Institutes of Health announce small-scale trials of two vaccines to 320 patients at high risk for AIDS.