Immunology and vaccination
Pasteur's first work on vaccine development was on chicken cholera. He received the bacteria samples (later called Pasteurella multocida after him) from Henry Toussaint. Being unable to conduct the experiments himself due to a stroke in 1868, Pasteur relied heavily on his assistants Emile Roux and Charles Chamberland. The work with chicken cholera was initiated in 1877, and by the next year, Roux was able to maintain a stable culture using broths. As documented later by Pasteur in his notebook in March 1880, in October 1879, being delayed in returning to the laboratory due to his daughter's wedding and ill health, he instructed Roux to start a new chicken cholera culture using bacteria from a culture that had sat since July. The two chickens inoculated with this new culture showed some symptoms of infection, but instead of the infections being fatal, as they usually were, the chickens recovered completely. After further incubation of the culture for an additional 8 days, Roux again inoculated the same two chickens. As was also noted by Pasteur in his notebook in March 1880, and contrary to some accounts, this time the chickens died. Thus, although the attenuated bacteria did not provide immunity, these experiments provided important clues as to how bacteria could be artificially attenuated in the laboratory. As a result, upon Pasteur's return to the laboratory, the focus of the research was directed at creating a vaccine through attenuation.
In February 1880, Pasteur presented his results to the French Academy of Sciences as "Sur les maladies virulentes et en particulier sur la maladie appelée vulgairement choléra des poules (On virulent diseases, and in particular on the disease commonly called chicken cholera)" and published it in the academy's journal (Comptes-Rendus hebdomadaires des séances de l'Académie des Sciences). He attributed that the bacteria were weakened by contact with oxygen. He explained that bacteria kept in sealed containers never lost their virulence, and only those exposed to air in culture media could be used as vaccine. Pasteur introduced the term "attenuation" for this weakening of virulence as he presented before the academy, saying:We can diminish the microbe's virulence by changing the mode of culturing. This is the crucial point of my subject. I ask the Academy not to criticize, for the time being, the confidence of my proceedings that permit me to determine the microbe's attenuation, in order to save the independence of my studies and to better assure their progress... [In conclusion] I would like to point out to the Academy two main consequences to the facts presented: the hope to culture all microbes and to find a vaccine for all infectious diseases that have repeatedly afflicted humanity, and are a major burden on agriculture and breeding of domestic animals.
In fact, Pasteur's vaccine against chicken cholera did not consistently produce immunity, and has subsequently been proven to be ineffective.
Following the results with chicken cholera, Pasteur eventually utilized the immunization method developed for chicken cholera to create a vaccine for anthrax, which affected cattle. In 1877, Pasteur had earlier directed his laboratory to culture the bacteria from the blood of infected animals, following the discovery of the bacterium by Robert Koch.
When animals were infected with the bacteria, anthrax occurred, proving that the bacteria was the cause of the disease. Many cattle were dying of anthrax in "cursed fields". Pasteur was told that sheep that died from anthrax were buried in the field. Pasteur thought that earthworms might have brought the bacteria to the surface. He found anthrax bacteria in earthworms' excrement, showing that he was correct. He told the farmers not to bury dead animals in the fields.
Pasteur's interest in creating a vaccine for anthrax was greatly stimulated when on 12 July 1880, Henri Bouley read before the French Academy of Sciences a report from Henry Toussaint, a veterinary surgeon, who was not a member of the academy. Toussaint had developed anthrax vaccine by killing the bacilli by heating at 55 °C for 10 minutes. He tested his vaccine on eight dogs and 11 sheep, half of which died after inoculation. It was not a great success. Upon hearing the news, Pasteur immediately wrote to the academy that he could not believe that dead vaccine would work and that Toussaint's claim "overturns all the ideas I had on viruses, vaccines, etc." Following Pasteur's criticism, Toussaint switched to carbolic acid (phenol) to kill anthrax bacilli and tested the vaccine on sheep in August 1880. Pasteur thought that this type of killed vaccine should not work because he believed that attenuated bacteria used up nutrients that the bacteria needed to grow. He thought oxidizing bacteria when sitting in culture broth for prolonged periods made them less virulent.
However, Pasteur's laboratory found that anthrax bacillus was not easily weakened by culturing in air as it formed spores – unlike chicken cholera bacillus. In early 1881, his laboratory discovered that growing anthrax bacilli at about 42 °C made them unable to produce spores, and he described this method in a speech to the French Academy of Sciences on 28 February. On 21 March, despite inconsistent results, he announced successful vaccination of sheep. To this news, veterinarian Hippolyte Rossignol proposed that the Société d'agriculture de Melun organize an experiment to test Pasteur's vaccine. Pasteur signed an agreement accepting the challenge on 28 April. Pasteur's assistants, Roux and Chamberland, who were assigned the task of conducting the trial, were concerned about the unreliability of the attenuated vaccine, and therefore Chamberland secretly prepared an alternative vaccine using chemical inactivation. Without divulging their method of preparing the vaccine to anyone but Pasteur, Roux and Chamberland performed the public experiment on May at Pouilly-le-Fort. 58 sheep, 2 goats and 10 cattle were used, half of which were given the vaccine on 5 and 17 May; while the other half was untreated. On 31 May, Roux and Chamberland next injected the animals with the fresh virulent culture of anthrax bacillus. The official result was observed and analyzed on 2 June in the presence of over 200 spectators, with Pasteur himself in attendance. The results were as Pasteur had bravely predicted: "I hypothesized that the six vaccinated cows would not become very ill, while the four unvaccinated cows would perish or at least become very ill." However, all vaccinated sheep and goats survived, while unvaccinated ones had died or were dying before the viewers. His report to the French Academy of Sciences on 13 June concludes:[By] looking at everything from the scientific point of view, the development of a vaccination against anthrax constitutes significant progress beyond the first vaccine developed by Jenner, since the latter had never been obtained experimentally.Pasteur did not directly disclose how he prepared the vaccines used at Pouilly-le-Fort. Although his report indicated it as a "live vaccine", his laboratory notebooks show that he actually used potassium dichromate-killed vaccine, as developed by Chamberland, quite similar to Toussaint's method.
The notion of a weak form of a disease causing immunity to the virulent version was not new; this had been known for a long time for smallpox. Inoculation with smallpox (variolation) was known to result in a much less severe disease, and greatly reduced mortality, in comparison with the naturally acquired disease. Edward Jenner had also studied vaccination using cowpox (vaccinia) to give cross-immunity to smallpox in the late 1790s, and by the early 1800s vaccination had spread to most of Europe.
The difference between smallpox vaccination and anthrax or chicken cholera vaccination was that the latter two disease organisms had been artificially weakened, so a naturally weak form of the disease organism did not need to be found. Pasteur's development of artificially weakened pathogens revolutionized work in infectious diseases, and he gave these artificially weakened diseases the generic name of "vaccines", in honour of Jenner's discovery.
In 1876, Robert Koch had shown that Bacillus anthracis caused anthrax. In his papers published between 1878 and 1880, Pasteur only mentioned Koch's work in a footnote. Koch met Pasteur at the Seventh International Medical Congress in 1881. A few months later, Koch wrote that Pasteur had used impure cultures and made errors. In 1882, Pasteur replied to Koch in a speech, to which Koch responded aggressively. Koch stated that Pasteur tested his vaccine on unsuitable animals and that Pasteur's research was not properly scientific. In 1882, Koch wrote "On the Anthrax Inoculation", in which he refuted several of Pasteur's conclusions about anthrax and criticized Pasteur for keeping his methods secret, jumping to conclusions, and being imprecise. In 1883, Pasteur wrote that he used cultures prepared in a similar way to his successful fermentation experiments and that Koch misinterpreted statistics and ignored Pasteur's work on silkworms.
In 1882, Pasteur sent his assistant Louis Thuillier to southern France because of an epizootic of swine erysipelas. Thuillier identified the bacillus that caused the disease in March 1883. Pasteur and Thuillier increased the bacillus's virulence after passing it through pigeons. Then they passed the bacillus through rabbits, weakening it and obtaining a vaccine. Pasteur and Thuillier incorrectly described the bacterium as a figure-eight shape. Roux described the bacterium as stick-shaped in 1884.
Pasteur's laboratory produced the first vaccine for rabies using a method developed by his assistant Roux, which involved growing the virus in rabbits, and then weakening it by drying the affected nerve tissue. The rabies vaccine was initially created by Emile Roux, a French doctor and a colleague of Pasteur, who had produced a killed vaccine using this method. Pasteur wrote that the vaccine had been tested in 50 dogs before its first human trial. This vaccine was used on 9-year-old Joseph Meister, on 6 July 1885, after the boy was badly mauled by a rabid dog. This was done at some personal risk for Pasteur, since he was not a licensed physician and could have faced prosecution for treating the boy. After consulting with physicians, he decided to go ahead with the treatment. Over 11 days, Meister received 13 inoculations, each inoculation using viruses that had been weakened for a shorter period of time. Three months later he examined Meister and found that he was in good health. Pasteur was hailed as a hero and the legal matter was not pursued. Analysis of his laboratory notebooks shows that Pasteur had treated two people before his vaccination of Meister. One survived but may not actually have had rabies, and the other died of rabies. Pasteur began treatment of Jean-Baptiste Jupille on 20 October 1885, and the treatment was successful. Later in 1885, people, including four children from the United States, went to Pasteur's laboratory to be inoculated. In 1886, he treated 350 people, of which only one developed rabies. The treatment's success laid the foundations for the manufacture of many other vaccines. The first of the Pasteur Institutes was also built on the basis of this achievement.
In The Story of San Michele, Axel Munthe writes of some risks Pasteur undertook in the rabies vaccine research:
Pasteur himself was absolutely fearless. Anxious to secure a sample of saliva straight from the jaws of a rabid dog, I once saw him with the glass tube held between his lips draw a few drops of the deadly saliva from the mouth of a rabid bull-dog, held on the table by two assistants, their hands protected by leather gloves.
Because of his study in germs, Pasteur encouraged doctors to sanitize their hands and equipment before surgery. Prior to this, few doctors or their assistants practiced these procedures. Ignaz Semmelweis and Joseph Lister had earlier practiced hand sanitizing in medical contexts in the 1860s.
