A Short History of Vaccination

EARLY DEVELOPMENTS

Attempts to “vaccinate” began long before Edward Jenner. While the precise origin of variolation remains unknown, it seems to have developed somewhere in Central Asia in the early part of the second millennium and then spread east to China and west to Turkey, Africa, and finally Europe.

In the 7th century, some Indian Buddhists drank snake venom in an attempt to become immune to its effect. They may have been inducing antitoxin-like immunity. In the 16th century, Brahmin Hindus in India practiced a form of variolation by introducing dried pus from smallpox pustules into the skin of a patient. Texts citing inoculation and variolation in 10th-century China make interesting reading but cannot be verified. However, 18th-century documentation of Chinese variolation. The Golden Mirror of Medicine , a medical text dated 1742, listed four forms of inoculation against smallpox practiced in China since 1695:

• The nose plugged with powdered scabs laid on cotton wool

• Powdered scabs blown into the nose

• The undergarments of an infected child put on a healthy child for several days

• A piece of cotton smeared with the contents of a vesicle and stuffed into the nose ^,

This text, endorsed by the Imperial Court, raised the status of variolation in China, which previously had been considered a folk remedy. Another Chinese text, published a century before Jenner, stated that white cow fleas were used for smallpox prevention. The fleas were ground into powder and made into pills.

Variolation was introduced into England by Lady Mary Wortley Montagu in 1721, after her return from Constantinople, where she lived for 2 years with her husband, the British Ambassador to the Ottoman Empire. She had been disfigured by smallpox earlier in life and her 20-year-old brother had died of the disease. While living in Turkey, she frequently observed variolation and wrote to a friend back home: “The small-pox, so fatal, and so general amongst us, is here entirely harmless, by the invention of engrafting, which is the term they give it…. Every year, thousands undergo this operation…they take the small-pox here by way of diversion, as they take the waters in other countries. There is no example of any one that has died of it….” So impressed was she that she had her own son variolated while still in Turkey. Dr. Charles Maitland, who performed the procedure on her son in Constantinople, later performed the first variolation in England in 1721 on Lady Montagu’s daughter. The treatment was effective, but results were erratic: 2–3% of persons treated died of smallpox contracted from the variolation itself.

The English medical community had previously learned of variolation in 1713 when Emanuel Timoni, MD, a graduate of Oxford University living in Turkey, sent a letter to the Royal Society about variolation. Giacomo Pilarino, MD, also reported Turkish variolation to the Royal Society in 1716. The reports did not seem significant, and the procedure was not adopted. Another hint of variolation was made by the Danish physician Thomas Bartholin of Copenhagen in 1675, who mentioned a “market” in Copenhagen where people went to buy the poxvirus from enterprising housewives. It is unclear whether these purchases were for the prevention of smallpox in healthy persons or for the treatment of persons already infected. Voltaire lauded the variolation of Circassian women to maintain their beauty in his Lettres Philosophiques in 1721.

Most intriguing, concurrent with Charles Maitland variolating Lady Montagu’s daughter in England (1721), variolation was practiced in America at the instigation of Cotton Mather who first learned of it from his African slave, Onesimus. Mather subsequently read about variolation in the Philosophical Transactions of the Royal Society of London , 1714–1716, which contained the aforementioned articles by Timoni and Pilarino. Smallpox was epidemic in Boston at that time, and Mather used the authority and prestige of his position to urge Boston-area physicians to consider the practice of variolation (letter, June 24, 1721). Physician response was negative, except for Dr. Zabdiel Boylston, who successfully inoculated his own 6-year-old son and two black slaves shortly thereafter. Six weeks later, Dr. Boylston variolated Mather’s son, Sammy, and publicized its success. Mather continued to harangue recalcitrant physicians and the public about variolation to the point where a grenade was thrown into his house in utter exasperation! ^,

Despite the known risks, George Washington felt compelled in the 1770s to order that Continental Army recruits undergo variolation against smallpox, to which the Americans were highly susceptible; the great majority of their English enemies were immune from early childhood exposure or from variolation. In the mid-18th century, several treatises were written on inoculation against measles as well; the Scottish physician Francis Home successfully inoculated humans against measles and published his results (1758).

In 1774 in Yetminster, England (Dorset County), a cattle breeder named Benjamin Jesty, himself immune to smallpox after contracting cowpox from his herd, deliberately inoculated his wife and two children with cowpox to avoid a smallpox epidemic. This was no spur-of-the-moment idea; like many country farmers in the area, he knew that dairymaids seemed to be protected from smallpox after they had contracted cowpox. He had considered the possibility of deliberately using the inoculation technique with cowpox for quite some time and acted only when there was an imminent threat of a smallpox outbreak. He took his wife and children to a nearby field where he knew he could find cattle with cowpox. He inoculated all three of them. His experiment succeeded; they were unaffected by the outbreak, and his sons were still immune 15 years later, when they were deliberately variolated with smallpox.

Jesty’s story is interesting; although neither a physician nor a scientist, he nevertheless reflected on the evidence of local dairymaids’ immunity to smallpox because of prior infection with cowpox and saw the principle involved: inoculation with one moderately harmless disease (cowpox) could provide protection against another, far more dangerous disease, smallpox. When Jesty’s neighbors learned that his wife had developed inflammation at the site of the inoculation and had to be treated by a physician because he had “vaccinated” her, they vehemently scorned him. Jesty retreated in the face of their disapproval and never attempted to publicize his experiment or to vaccinate anyone else. Truly, Jesty’s actions constituted the first known real vaccination —the use of cowpox to protect against smallpox.

Some 30 years later, through the intercession of an enthusiastic vaccinator named Rev. Andrew Bell, Jesty was invited to London in 1805 by the Original Vaccine Pock Institute to tell the story of his 1774 “experiment” before the Institute’s examiners. At the end of the visit, a public statement in the Edinburgh Medical & Surgical Journal recognized Jesty’s cowpox vaccination. They commissioned his portrait as well, which was hung in the Institute. ^, This was a vindication of sorts, but he did not share in the significant monetary award that Jenner received. When Jesty died in 1816, his wife made certain that his tombstone recorded for all posterity his central role in that great endeavor (see frontispiece).

Nevertheless, Jenner’s work with cowpox vaccination still holds title to the first scientific attempt to control an infectious disease on a large scale by means other than transmitting the disease itself.

Cowpox was not widespread. It appeared sporadically in certain rural English counties. The local wisdom that persons who contracted cowpox “did not take the smallpox” was not widely known. Jenner knew it because he had been an apothecary apprentice in Chipping Sodbury in 1768, where a milkmaid told him about it. Indeed, he discussed the possible association between the two with John Hunter, with whom he studied in London from 1770 to 1773. For unknown reasons, Jenner did not return to the subject of cowpox/smallpox until 1796. His first manuscript to the Royal Society on vaccination was rejected because his experiment involved only one person, not enough to establish a principle. ^, Within 2 years, he expanded his studies and proved that cowpox could be passed directly from one person to another, thereby providing “large-scale” inoculation against smallpox without depending on the sporadic outbreaks of natural cowpox. Jenner self-published his results in Variolae Vaccinae in 1798. This publication brought to the attention of the entire medical community the merits of inoculation with the relatively obscure animal disease, cowpox, to prevent one of humankind’s deadliest scourges. Fortunately for the world, Jenner had the connections that Jesty did not, and vaccination rapidly replaced variolation. Jenner is not the originator of the term vaccination ; that honor belongs to his friend Richard Dunning, who used it in 1800. ^, By 1810, Jenner realized that smallpox immunity by vaccination was not lifelong, but he did not know why.

Curiously, the vaccinia virus used in current smallpox vaccine is not the cowpox virus that Jenner used. Vaccinia, cowpox, and variola are all related orthopox viruses, suspected to have been derived from a common ancestor. S. Monkton Copeman gave considerable attention to this issue in the Milroy Lectures of 1898, which make fascinating reading. He documents the many ways, times, and methods by which cows were inoculated to keep the supply of cowpox intact, further adding to the confusion of when and how vaccinia replaced cowpox. It has been suggested that vaccinia may have originated in a now extinct horsepox. What is clear is that the exact origin of vaccinia remains unknown, as does how it became substituted for cowpox.

For the first several decades of the 19th century, arm-to-arm transfer was the primary method of human vaccination. Other recognized diseases, such as syphilis and tuberculosis, were occasionally transmitted along with the “cowpox” virus, so a search began to find an alternative way to vaccinate and to ensure a steady supply of cowpox vaccine. The concept of “passages” of the immunizing agent (transmission from one human or animal to another) was well known, and in 1836, Edward Ballard argued for choosing new strains of cowpox for vaccination because the old strains were too weak (too attenuated) from so many arm-to-arm passages. He recommended that the lymph (vesicle fluid) be passed back through a calf to regain strength.

According to Ballard, the idea to use animals to propagate cowpox vaccine, as opposed to human arm-to-arm propagation, was first practiced in Naples, Italy, in 1805. Troja used vaccine virus derived from humans to inoculate cows and then used the lymph from the cows’ pocks to vaccinate humans. This was referred to as retrovaccination . Troja’s successor, Galbiati, stated that he used bovine lymph specifically to avoid transmission of other human diseases. By 1842, a third Italian, Negri, gave up the practice of retrovaccination entirely. He started what was called animal vaccination , that is, inoculating from one cow to another cow to keep a steady supply of cowpox lymph. But the initial virus source that he inoculated into the cow was from a human! When a natural outbreak of cowpox occurred in Calabria, Negri switched to that source for his lymph, but because he was running a commercial activity, he bought a third “cowpox strain” from London. Its origin was questionable, but Negri used it anyway, from 1858 onward. By mid-century, arm-to-arm vaccination was essentially replaced by animal vaccination, but it is easy to understand why the origin of the vaccinia virus is difficult to trace. ^,

In 1864, a French physician named Lanoix studied animal vaccination in Naples and brought to France a calf inoculated by Negri. He and Chambon set up a French business for production of calf-to-calf lymph vaccine for humans. ^{, ,} The French government became interested and ordered a study of animal vaccination. The lymph used in the experiments was obtained from Negri in Italy but was really from the London cowpox lymph of questionable origin. By chance, there were two outbreaks of cowpox in France in 1866. Lanoix and Chambon collected cowpox from both, mixed them together, and used the mixture to produce their vaccine. ^, From these confused beginnings, animal vaccination spread rapidly throughout the continent.

Following Robert Koch’s recommendation, German scientists began to use glycerin to kill bacteria and to preserve the lymph. This generated a ready supply of a stable calf lymph of consistent potency. By the end of the 1890s, the use of glycerinated calves’ lymph was standard everywhere, and both arm-to-arm vaccination and unglycerinated animal vaccination were abandoned.

Louis Pasteur and the Age of Vaccination

Louis Pasteur’s ( Fig. 1.2 ) work on the attenuation of the chicken cholera bacterium in the late 1870s was the first major advance after Jenner’s Variolae Vaccinae . Pasteur drew on concepts that had been developing for at least 40 years: attenuation; modification through passage; renewed virulence; and, most important, the need to replace person-to-person (or animal-to-animal) vaccination with something safer, consistent, and less likely to transmit other diseases.

The popular story, that Pasteur had a “eureka” moment when he noticed that a chicken cholera culture ( Pasteurella multocida ) left exposed to air during a long holiday period provided immunity when challenged, is considerably more complex than originally told. From the moment he obtained the chicken cholera culture from Henry Toussaint in October 1878, Pasteur spoke of wanting to manipulate it to make a vaccine. The culture was virulent; he killed many chickens trying to keep it alive through passage from chicken to chicken. By January 1879, he found that he could keep the microbe alive in a culture of chicken bouillon. Chickens inoculated from this bouillon culture died, so it had retained the virulence of his original sample. Next, he prepared a bouillon culture using inflamed chicken muscle tissue from the site of inoculation. He noted that this infected muscle tissue culture did not develop normally as the other cultures did; it became acidic. He fed the chickens bread soaked in the first bouillon culture and then fed them bread soaked in the muscle bouillon. They became sick but survived. He then challenged them with virulent organisms, and they lived, so he thought he had a vaccine. After the challenge, the chickens sickened again but again survived and eventually returned to health. By March 1879, Pasteur realized that he had attained “resistance” to disease; he also knew he hadn’t yet found a product that could be safely used as a vaccine. For the next several months, Pasteur subjected the chicken cholera microbe to various conditions, for example, in a vacuum, exposed to air, and various intervals before inoculation. But it was mainly the chickens that had eaten the bread soaked in acidic bouillon that survived.

Pasteur left on vacation at the end of July 1879; he returned in October and again took up his chicken cholera experiments, focusing on the cultures that had become acidic because they seemed to be the ones that conferred immunity. He tinkered with the amount of time the microbe remained in the acidic cultures. In December 1879, he noted that two cultures made from a chicken that died of a prevacation inoculation were the only cultures that turned acidic. He focused on them for his vaccine, although he still had many problems with illness in the chickens. By the middle of January 1880, Pasteur realized that the diminution of virulence came from leaving the microbe for a long time in an acid culture.

Pasteur’s definitive test was on January 22, 1880. With highly virulent chicken cholera, he inoculated 19 naïve chickens and 8 that had been previously immunized twice with his acidic cultures. All eight of the previously immunized chickens lived. Most of the naïve candidates did not. So Pasteur’s eureka moment was really the result of months of intensive, deliberate research. He presented his results at the Académie des Sciences on February 8, 1880, and at the Académie de Médicine on February 10, 1880, announcing that he had achieved successful vaccination against chicken cholera, but he did not reveal his technique. He continued to refine the technique and tried to increase the stability of the product. Finally, in October 1880, he published at least part of his method for preparing the vaccine. ^, Pasteur’s chicken cholera vaccine harkened back to the classic variolation technique, which had used a weakened form of smallpox to inoculate against smallpox. Therefore, the modern concept of vaccination, involving the development of vaccines in the laboratory and using the same agent that caused the disease, was truly introduced with Pasteur’s chicken cholera vaccine, 5 years before the famous vaccination of Joseph Meister against rabies. Ironically, chicken cholera vaccine was never a success; there were frequent vaccine failures. Pasteur was lauded, but the vaccine was eventually discontinued.

Pasteur’s research on anthrax began in 1877 and overlapped his work on chicken cholera. Casimir Davaine had seen the anthrax bacillus in 1850, and postulated it as the cause of anthrax, ^, but Koch was the first to obtain pure cultures of anthrax bacillus and to describe its capability to survive indefinitely in the form of spores. He transmitted it to several laboratory animals and proved that there was a causal relationship between this bacillus and the disease anthrax.

Pasteur knew of Davaine’s and Koch’s work and that of the veterinarian Toussaint ( Fig. 1.3 ). Indeed, he was in a neck-and-neck competition with Toussaint to develop an anthrax vaccine. Toussaint published two articles in July 1880 in the Comptes Rendus de l’Académie des Sciences on a live anthrax vaccine that he developed and tested, which induced immunity against anthrax. ^, He had started his anthrax experiments in May 1880, stating that he was inspired to do so after hearing Pasteur in February 1880 describe his chicken cholera vaccine experiment, although Pasteur had not yet revealed his method. Toussaint used the blood of bovines that had died of anthrax, noting that the fluid was full of bacteria, which others thought was extraneous material but Toussaint thought was the causative agent of anthrax. He first attempted to simply “filter” the blood and use it as a vaccine but quickly realized that the bacteria could pass through the filter. He then decided to follow the procedure that Davaine described: to heat the blood for 10 minutes at 55°C or subject the blood to diluted phenol. He used both of these techniques, and a variation on filtering in which he used 12 filters, to produce attenuated vaccines that he injected in rabbits, sheep, and young dogs. The animals were protected by all three methods, although Toussaint initially thought that the heat-treated was the best.

Toussaint’s articles caused quite a stir at the Académie; Pasteur and others challenged their validity and Toussaint was compelled to not only reveal his methods, but also to conduct experiments to prove his claim. On July 28, while still in Toulouse, he took fresh blood from a sheep dying of anthrax and prepared his vaccine in two lots, one with 1% phenol and the other with 1.5%. Both lots were filtered, although by different means. Neither lot was heat-treated . He left for Paris with his two lots of “vaccine,” where his experiment continued at Vincennes and Alfort under the watchful eyes of several researchers. On August 8, Toussaint inoculated 20 sheep with the first solution (1% and filter papers); 4 of them died very quickly, but the other 16 survived. On August 22, six new sheep were inoculated with material from the second lot (1.5% phenol and rudimentary filtration), and all survived without illness. All 22 sheep were then subjected to challenge with virulent anthrax and all survived. With one injection of a (partially) live vaccine attenuated by filtration and phenol acid, Toussaint had achieved immunity to anthrax!

It should be reemphasized that Pasteur did not reveal his method for making chicken cholera vaccine until October 1880. Toussaint’s vaccine work was original; it was his own, not Pasteur’s. He indeed induced immunity to anthrax, his was the first anthrax vaccine. Eventually, he received the Prix Vaillant and the Légion d’Honneur for this work. ^, Toussaint came down with a debilitating neurologic disease in 1881, which prevented him from pursuing his claim as the originator of the first anthrax vaccine. His health continued to decline, and he died in 1890 at age 43. ^,

The following spring, Pasteur announced the first public controlled experiment of anthrax vaccination at Pouilly-le-Fort, May 5,1881. It was initiated by Pasteur in an effort to silence his many critics and to gain recognition for his own anthrax vaccine. Pasteur inoculated 24 sheep, 1 goat, and 6 cows with attenuated anthrax bacilli. On May 17, these same animals were inoculated again with more virulent but still attenuated anthrax bacilli. At the same time, 24 sheep, 1 goat, and 4 cows were kept as control animals and given no inoculations. On May 31, both groups were inoculated with virulent anthrax from spores that Pasteur had kept in his laboratory since 1877.

By June 2, 21 of the nonvaccinated sheep and the nonvaccinated goat were dead. Two more nonvaccinated sheep died before the spectators’ eyes, and the last one died before day’s end. All vaccinated sheep, the vaccinated goat, and the six cows remained healthy. (The nonvaccinated cows did not die but showed clear evidence of having contracted anthrax. Their size perhaps had saved them.) At the end of this experiment, the triumphant Pasteur wrote that he had shown that vaccines could be made that were cultivatable at will by a method that could be generalized, since he had already used the method previously to create a vaccine against chicken cholera. His experiment represented a considerable advancement over Jennerian vaccination, which had not been subjected to the rigors of a controlled experiment.

It has since been documented that Pasteur’s results with chicken cholera and anthrax were not as clear-cut as previously thought. Pasteur deliberately withheld critical data (conflicting information on the degree of protection of the vaccine) in his communications to the Académie de Médicine. However, this in no way detracts from the significance of his findings, which proved that one could “create” standardized, reproducible vaccines. Pasteur’s experiments with chicken cholera and anthrax ^, announced to the world that a new, scientific era in vaccination had begun.

By the time the rabies vaccine was first administered to humans in 1885, the general public and the scientific community were well aware of the “new vaccination,” but only in relation to animals. The reaction when Joseph Meister and Jean Baptiste Jupille became the first humans to be vaccinated against rabies was predictable: outrage. Meister, a 9-year-old boy from Alsace, had been bitten 14 times on the hands and thighs and arrived in Paris some 60 hours after he had been attacked by a rabid dog. The physician working with Pasteur, Dr. Joseph Grancher, was convinced that Meister would die of rabies if left untreated and, therefore, the attempt to vaccinate was justified to save his life. Meister was vaccinated in the same manner that Pasteur was using in his experiments to protect animals (especially dogs) against rabies: with a series of progressively less dried and, therefore, more virulent rabbit spinal cords obtained from rabbits that had died of rabies after having received “fixed virus” injections of rabies virus. A couple of months later, Jean Baptiste Jupille, a 14-year-old from the Jura region of France, arrived 6 days after having been bitten multiple times. (He had fought off a rabid dog that had attacked some younger children.) He was given the same course of treatment that Meister received. They both survived.

That Pasteur had deliberately introduced a deadly agent into a human left people aghast. The fact that the rabies virus had been attenuated did not appease the general public or many in the medical community; the cases of rabies that occasionally occurred in subsequent vaccinees were attributed to the vaccine and were viewed as medical murders. Even Émile Roux, one of Pasteur’s staunchest allies and a collaborator in the rabies experiments, was appalled at the vaccination of Meister. He thought it was unjustified by the experiments conducted up to that point.

An examination of Pasteur’s laboratory notebooks indicates that Roux was right to object. The notebooks tell us that, shortly before vaccinating Meister, in May and June of that same year, Pasteur had seen and recommended vaccination for two other people in local hospitals, each of whom had been admitted with the presumed diagnosis of rabies. The first, an adult, was admitted with an uncertain diagnosis of rabies. The second case was a young girl, 11 years old, who had been bitten by a rabid dog on the lip, remained untreated, and was admitted to a hospital a month later with frank rabies. In both cases, Pasteur used a rabies vaccine made of an emulsion of desiccated spinal cord from a rabid rabbit. Up to this point, he had never published anything about using spinal cords as a vaccine, and in fact, had not yet successfully protected any animal from rabies with such a vaccine. The first patient, the adult whose diagnosis was uncertain, received only one–unauthorized–dose of the vaccine before his doctor forbade the administration of further doses. This patient lived. The young girl, whose rabies diagnosis was not in doubt, received two injections of the same vaccine on her first day in the hospital. When Pasteur and his nephew Adrian Loir arrived the next morning to give the third injection, the young girl died before she could receive it. The death was almost certainly from rabies and not from the two injections she had received. These two cases were never published by Pasteur but were subsequently found in his laboratory notebooks.

Roux left Pasteur’s laboratory in protest after Meister’s vaccination and did not return until the summer of 1886, after several dozen people who had been bitten by rabid animals had been successfully vaccinated. ^{, , ,} Ultimately, hundreds were saved from rabies, many more than had died despite the vaccination (presumably from rabies contracted from the bites). That did not lessen the strenuous opposition to rabies vaccination in humans nor the belief by many that the vaccination itself caused the deaths. After all, only 45 years earlier, once Jenner’s vaccination had been accepted, variolation had been made a felony in England for the very same reason: it introduced a deadly live virus into humans (as opposed to cowpox, which was not deadly). Grancher, the physician who administered the rabies vaccine to Meister and many others, was one of Pasteur’s staunchest supporters and was invaluable in defending Pasteur before the Académie de Médicine and against recalcitrant physicians. Despite the opposition, and thanks to Grancher and other supporters, Pasteur soon became a worldwide medical hero.

The next major step in vaccine development took place in the United States. It involved a new concept that was equally important: killed vaccines. In 1886, Daniel Elmer Salmon ( Fig. 1.4 ) and Theobald Smith ( Fig. 1.5 ) published their work on a killed hog cholera “virus” vaccine. ^, The heated suspension of organisms immunized pigeons against the disease. The vaccine they developed was actually a bacterial vaccine against a cholera-like salmonellosis, but the term virus in the latter half of the 19th century did not have the specific meaning it has today; there was confusion about what pathogens could pass through filters. Their report demonstrated that the ideas of live and killed vaccines developed almost simultaneously. This seminal work of Salmon and Smith bore fruit for humans 15 years later.

Their competitors in the development of a killed vaccine were Charles Chamberland and Roux from Pasteur’s laboratory, who reported on the same topic in December 1887, some 16 months after Salmon and Smith’s original paper. In 1888, Salmon read a paper before the American Association for the Advancement of Science (AAAS) defending their 1886 article and their priority in developing the first killed vaccine. However, the Institut Pasteur had just been established in 1887; Pasteur was at the height of his fame and worldwide prestige thanks to the rabies vaccine. Not surprisingly, Salmon and Smith, working for the U.S. Department of Agriculture, saw their claim lost in the aura surrounding Pasteur and his associates. Thus, even 100 years ago, the Institut Pasteur and the U.S. government were involved in disputations about discovery rights, similar to the late 20th-century controversy about who first isolated the human immune deficiency virus: Luc Montagnier at the Institut Pasteur or Robert Gallo at the National Institutes of Health.

Killed vaccines for typhoid, plague, and cholera followed Salmon and Smith’s research. Richard Pfeiffer and Wilhelm Kolle in Germany and Almroth Wright in England worked independently on killed typhoid vaccines. To this day, the debate continues about who inoculated the first human with killed typhoid vaccine. In truth, all three deserve credit because it is now clear that several groups were working on typhoid vaccine at that time.

Shibasaburo Kitasato and Alexandre Yersin, each working independently, discovered the causative bacillus of the plague in 1894, Yersinia pestis (called Pasteurella pestis until 1970). ^{, , ,} With Albert Calmette and Amédée Borelle, Yersin developed a killed plague vaccine for animals, but it was Waldemar Haffkine who took up the task of developing a vaccine against human plague. ^, Haffkine was in India working on cholera vaccine when bubonic plague broke out in Bombay. He switched to studies of plague immunization and was himself the first to be injected with his new killed plague vaccine. More than 8000 people were then vaccinated within a few weeks. For a while, Haffkine was a hero. However, the Mulkowal incident in 1902 when 19 people died from contaminated plague vaccine resulted in Haffkine’s removal from his post by the Indian government. The contamination (with tetanus bacillus) does not seem to have been his fault. Nevertheless, his scientific career and reputation were severely damaged; he never fully recovered from the incident and retired early from science at age 55. Later, with the wisdom of hindsight, the Indian government renamed the Plague Research Laboratory where he had worked The Haffkine Institute . Perhaps as important as his development of the plague vaccine was Haffkine’s contribution to the literature on the proper way to conduct controlled field trials.

John Snow had shown that cholera was transmitted by contaminated water in 1848, although he did not know the identity of the contaminant. Koch supplied the answer, when he isolated Vibrio cholerae as the causal organism in 1883. Early attempts at a cholera vaccine were made by Jaime Ferrán, Pasteur’s pupil, and by Haffkine. Both used live cultures and both vaccines were rejected because of severe reactions. Kolle developed a heat-killed human cholera vaccine in 1896. ^, He grew the vibrios in agar, suspended them in saline solution, heated them at 50°C for a few minutes (later changed to 56°C for 1 hour), and then added 0.5% phenol.

In parallel with vaccine research, important work on immunity was pursued at the end of the 19th century. Elie Metchnikoff, another Pasteur protégé, reported his theory of cellular immunity in 1884. ^, He named the cells that ingested and destroyed invading microorganisms and other foreign bodies phagocytes . Although he did not understand the role of serum and plasma in immunity at this early date, his work was truly pioneering.

In 1888, Roux and Yersin showed that the diphtheria bacillus produced a powerful toxin . ^, Two years later, Emil von Behring and Kitasato, working in Koch’s laboratory in Berlin, followed up on early work by Karl Fraenkel; they showed the presence of powerful antitoxins in the serum of animals previously injected with low doses of tetanus or diphtheria toxins. The antitoxin neutralized diphtheria or tetanus toxin in culture. Further experiments showed that the antitoxin protected animals challenged with the tetanus or diphtheria bacillus. Although he did not use the term, what von Behring had found in the serum of animals previously injected with diphtheria or tetanus toxins were antibodies . It was Paul Ehrlich, also working in Koch’s laboratory, who first referred to these antitoxins as antibodies—“antikorps.”

Progress occurred rapidly after these reports; the first child was treated with diphtheria antitoxin 1 year later, December 1891. Shortly thereafter, commercial production of diphtheria antitoxin began. von Behring referred to the rabbit serum that contained the antitoxin as “immune serum.” Soon, the process of inoculating with the immune serum that contained tetanus or diphtheria antitoxin was referred to for the first time as immunization . ^,

Ehrlich’s receptor theory of immunity, which he referred to as the “side-chain theory,” made a strong contribution to vaccine development. When it was first developed in 1897, the theory was used primarily to explain toxin–antitoxin interactions and subsequently the relationship between antigens and antibodies. It became one of the cornerstones of 20th-century immunology. Ehrlich’s other major contribution was to point out the difference between active and passive immunity. ^,

The last decade of the 19th century produced remarkable advancements from remarkable men. von Behring was awarded the first Nobel Prize in Medicine (1901), Koch received it in 1905, and Ehrlich and Metchnikoff shared the Nobel in 1908.

FIRST HALF OF THE 20TH CENTURY

At the beginning of the 20th century, five human vaccines were in use: Jenner’s original smallpox vaccine, Pasteur’s rabies vaccine (both containing live virus) and three bacterial vaccines: typhoid, cholera, and plague (all killed). In addition, immunization with diphtheria or tetanus antitoxin was an accepted practice. The year 1900 also saw the end of arm-to-arm lymph inoculation as a vehicle for smallpox vaccination. It was replaced by the use of glycerinated calf lymph in 1898. Most of the fundamental concepts of vaccinology had been introduced by the end of the 19th century; the early 20th century would bring refinements. Not until the advent of cell culture 50 years later would the field again become so dramatically fertile ( Table 1.1 ).

Wright proposed mass immunization of British troops with killed typhoid vaccine during the Boer War (1899), but because of opposition to adverse reactions, he was able to vaccinate only 14,000 volunteers. Opposition ran so high that consignments of vaccine were jettisoned from transport ships in Southampton. The result was catastrophic: more than 58,000 cases of typhoid and 9000 deaths in the British Army. A bitter battle about the merits of the vaccine was waged in the British Medical Journal between Wright and the statistician Karl Pearson. Ultimately, at Wright’s insistence, the War Board initiated a broad-based trial that showed the overwhelming effectiveness of the vaccine. Wright was then knighted. By the beginning of World War I in 1914, general typhoid vaccination was conducted in the British Army, although it was still not mandatory. ^{, ,}

During the first few decades of the 20th century the use of “bacterins” as human vaccines came into use. Bacterins consisted of killed bacteria (antigens) that were injected parenterally to produce active immunization. Sometimes they were combined with immune serum to become “serobacterins”—the serum would provide short-term immunity before the killed bacterial antigens kicked in for long-term immunity. The concept was an outgrowth of Metchnikoff’s theory of phagocytosis and Wright’s work on opsonins. It was thought that bacterins worked because opsonins induced by the antigens prepared invading bacteria for phagocytosis. Most bacterins were prepared and sold without clinical trials. In its 1908 catalog, HK Mulford Company, a forerunner to Merck, listed nine bacterins that it sold, including those for gonorrhea, typhoid, pneumococcal disease, and streptococcal disease. Bacterins for humans fell into disuse by the late 1930s as more stringent licensing requirements were imposed by the federal government. ^, Bacterins are still used as targeted vaccines for specific herds of animals.

In the early 20th century, the chemical inactivation of diphtheria and other bacterial toxins led to the development of the first toxoids : diphtheria and tetanus. Here again, Theobald Smith had a significant role. In 1907, he determined that toxoids provided immunity in guinea pigs. In a 1909 report on long-lasting immunity against diphtheria in guinea pigs immunized with toxoid, he suggested that toxoids should be considered for humans. ^,

In 1923, Alexander Glenny and Barbara Hopkins showed that diphtheria toxin could be transformed into a toxoid by formalin. The discovery came about when the diphtheria toxin containers were cleaned with formalin (they were too large to be autoclaved). The residual formalin in the vats rendered the subsequent batch of toxin so weak that 1000 times the normal dose did not kill the guinea pigs. Although this toxoid was certainly safer than the toxin, it could be administered only in conjunction with antitoxin. In that same year, Gaston Ramon developed a diphtheria toxoid that could be used on its own (i.e., without antitoxin) by adding formalin and incubating the mixture at 37°C for several weeks. Ramon and Christian Zoeller used a tetanus toxoid developed in the same manner for the first human vaccinations against tetanus in 1926. ^,

The vaccine against tuberculosis, bacille Calmette-Guérin (BCG), was the first live vaccine for humans to be produced since Pasteur’s rabies vaccine in 1885. Calmette was a protégé of Emile Roux and founder of the Pasteur Institutes at Lille and in Indochina. In 1906, Calmette and Camille Guérin, a veterinarian, started subculturing a strain of mycobacteria obtained from a bovine, which they perhaps thought was Mycobacterium tuberculosis but was in reality Mycobacterium bovis . They originally focused on producing a serotherapy, along the lines of von Behring’s antidiphtheria serotherapy, but quickly realized they could not easily inhibit the pathogenicity of the bacillus. That began their pursuit of a vaccine. After 13 years of attenuation by 230 passages in beef bile, potatoes, and glycerol, this strain eventually became the BCG strain. In total, Calmette and Guérin spent more than 20 years trying to understand the mechanism of infection of tuberculosis. Clinical trials in children began in 1921, and the vaccine became available for human use in 1927. ^, Because the original vaccine strain was sent to numerous laboratories around the world, each of which then produced its own variation of BCG vaccine, standardization has proved difficult. Despite the existence of more than a dozen major BCG vaccine strains that vary widely in strength, BCG remains an effective, if imperfect, vaccine against tuberculosis in children.

In 1931, E.W. Goodpasture introduced the use of the chorioallantoic membrane of the fertile hen’s egg as a medium for growing viruses. ^, This was a major advance because until then it was thought that human viruses could be grown only in animals such as ferrets and mice. Ferrets were expensive, and mouse brain could produce allergic brain encephalitis. The chick embryo proved to be a cheaper, safer medium for the cultivation of viruses. Earlier, in the second Milroy Lecture of 1898, Copeman described conducting an experiment using hens’ eggs—successfully—to grow vaccinia virus for the production of smallpox vaccine. ^,

Yellow fever virus was isolated in 1927 by two independent groups: researchers at the Rockefeller Foundation working in Nigeria, who isolated the Asibi strain ; and researchers at the Pasteur Institute in Senegal, who isolated the French strain. ^, The French strain was given to various research groups for study. In 1928, A.W. Sellards at the Harvard Medical School began collaborative research on the French strain with Jean Laigret at the Pasteur Institute in Senegal. Max Theiler, working for Sellards at Harvard, developed an animal model to study the virus. Using passage in mouse brain, others were able to “fix” the neurovirulence of the strain, which then was used as a vaccine. This French strain yellow fever vaccine from Theiler’s work at Harvard was a live vaccine derived from mouse brain passage. Sellards and Laigret intended to do human trials on yellow fever vaccine at the Institut Pasteur in Paris, but Roux, who was then the director, refused to allow human trials with the murine virus. He thought it was too dangerous. Eventually it was used in humans without immune serum by Sellards and Laigret in 1932. However, owing to the strain’s passage through mouse brain tissue, the neurovirulence of the French strain did indeed present grave dangers.

Theiler subsequently left Harvard to join the Rockefeller Institute and attempted to develop a more attenuated vaccine, using the Asibi strain. Theiler and Hugo Smith developed the 17D strain from Asibi in fertile hen’s eggs chorioallantois per Goodpasture’s method. Although the French strain was highly effective, the 17D strain was both effective and much safer. ^{, ,} The French strain certainly saved many lives, especially in French West Africa, where it was used extensively. It remained in production (in modified form) until 1982; however, safety concerns about mouse brain tissue overrode its proven efficacy and 17D won out as the vaccine strain of choice. For this work, Theiler was awarded the Nobel Prize in 1951.

Wilson Smith, Christopher Andrewes, and Patrick Laidlaw isolated human influenza A virus in ferrets in 1933. They followed the technique outlined by Richard Shope of the Rockefeller Institute when he isolated the swine influenza virus in pigs in 1931. Within 5 years, Smith’s group and Shope were able to show that swine influenza virus was a surviving virus from the great influenza pandemic of 1918. ^, Frank Horsfall, Alice Chenoweth, and colleagues developed a live influenza virus vaccine in mouse lung tissue in 1936. ^, Chenoweth claimed that it became inactivated or nonreplicating when it was administered parenterally. ^, That same year, 1936, saw the development of two influenza A vaccines grown in embryonated eggs, one (live) by Wilson Smith and the other (killed, whole virus) by Thomas Francis and Thomas Magill. ^, Even though these two vaccines were considered safer because they were developed in embryonated eggs, Chenoweth’s mouse lung vaccine had contained a higher virus yield and was the first to demonstrate true protection in humans, albeit transient.

In 1937, Anatol Smorodintsev and colleagues in the Soviet Union administered the Wilson Smith strain to humans by the intranasal route, using doses that were lethal when given to mice. This is considered the first live human influenza virus vaccine, and although it would not receive a passing grade by today’s standards (20% of vaccinees developed febrile influenza), it absolutely demonstrated the role of the virus in the development of influenza. ^,

Frank Burnet and D.R. Bull showed in the early 1940s that live attenuated influenza virus could be produced in embryonated eggs but also that the resultant virus mutated rapidly. Therefore, the vaccines that were produced were not consistently attenuated and often produced disease. ^, By contrast, the Francis and Magill killed, whole-cell influenza A vaccine did not have the problem of mutated viruses and did not produce disease.

In 1940, Francis and Magill independently isolated influenza B; at this point, it was recognized that at least three strains of influenza were circulating at the same time. Francis developed a killed (formaldehyde), trivalent (2As, 1B) vaccine that was mass-produced for the U.S. military in World War II. This conferred a certain “legitimacy” to killed influenza vaccine, as the military did not want to be concerned about “down time” from disease frequently associated with live influenza vaccine. During this same period, Burnet developed a live aerosolized influenza vaccine, but the influenza season had already begun, so an efficacy trial was inconclusive. By then, the killed vaccine of Francis had been very successful during the war. The Australian government denied Burnet permission to continue trials of a live vaccine as too risky. ^{, , ,} Except for the Soviet Union, which continued to use live vaccine, killed influenza vaccine became the standard until the 1990s (see subsequent text).

During the 1947 flu season, the influenza vaccine did not protect, definitively proving the concept of antigenic variation of the virus strain from year to year, which had first been proposed by Magill and Francis in 1936. The term itself, “antigenic drift,” was introduced by Burnet in 1955 in his Principles of Animal Virology .

Many attempts were made to develop vaccines against rickettsiae once Charles Nicolle had discovered in 1909 that they were the cause of typhus . The first truly successful typhus vaccine was developed in 1938 by Herald Cox, who used the yolk sac of the chick embryo to grow Rickettsia rickettsii . Cox was working on Rocky Mountain spotted fever at the time, but once he found a method to cultivate the rickettsia, killed vaccines for typhus and Q fever quickly followed. There was a heavy demand for the typhus vaccine during World War II. ^{, ,}

Jules Bordet and Octave Gengou first observed the causal agent of pertussis in 1900 and cultivated it by 1906. ^, Several vaccines were tested in small trials. Thorvald Madsen later carried out the first controlled clinical trials of a pertussis vaccine (i.e., whole killed organisms) on the Faeroe Islands in 1923–1924 and in 1929. ^, During the 1923–1924 epidemic, Madsen reported that the vaccine did not prevent disease but greatly reduced mortality and severity of illness among vaccinated persons. By the 1929 epidemic, the vaccine had been considerably improved but still did not prevent disease. In the 1930s, Pearl Kendrick and Grace Eldering ( Figs. 1.6 and 1.7 ), working for the Michigan Department of Health, improved the yield of the Bordet-Gengou growth medium and developed a killed (thimerosal) vaccine that they successfully tested in more than 1500 children. Only 4 of 712 vaccinees developed mild cases of whooping cough. They recruited the help of Eleanor Roosevelt to gain additional funds for further research and by 1940, their vaccine was distributed throughout the United States. The American Academy of Pediatrics approved the vaccine in 1943 and the American Medical Association in 1944. Several whole-cell pertussis vaccines were in use by the late 1940s. ^, The first combination vaccine, DTP (diphtheria, tetanus, pertussis) became available in 1948.