As a new generation of scientists looked back on the golden age of bac- teriology, their enthusiasm was tempered by the realization that microbe hunting did not in itself lead to the cure of disease. A re-evaluation of the factors that determined the balance between health and disease involved rejecting too narrow a bacteriological focus and working towards an understanding of human physiological responses to microbial agents. Certainly, the observation that surviving one attack of a particular disease provided protection from subsequent attacks was not new. This was the basis of the immunity earned in exchange for submitting to the risks
of smallpox inoculation or vaccination. The Latin term ‘‘immunity’’ orig- inally referred to an ‘‘exemption’’ in the legal sense. Since the intro- duction of Jennerian vaccination, it was clear that protective vaccines took advantage of the body’s own defense mechanisms, but the modern era of immunization began in the 1880s when Louis Pasteur proved that it was possible to attenuate pathogenic microbes and create speciﬁc vaccines in the laboratory. Building on the work of Louis Pasteur and Robert Koch, Shibasaburo Kitasato, Emil von Behring, and Paul Ehrlich developed new forms of treatment known as serum therapy and chemotherapy.
Coming from a family with many children and limited resources,
Emil Adolf von Behring (1854–1917) attended the Army Medical College in Berlin in exchange for 10 years of service in the Prussian Army. Military medicine provided an important route into the pro- fession for many men of modest means. After working as an assistant to Robert Koch at the Institute for Infectious Diseases, Behring held professorships at Halle and Marburg. Friedrich Althoff (1839–1908), one of the leading ofﬁcers of the Prussian Ministry of Education and Cultural Affairs, played a major role in advancing Behring’s career. When Behring became Director of the Institute of Hygiene at Marburg, he divided the Institute into two departments, a Research Department for Experimental Therapy and a Teaching Department for Hygiene and Bacteriology. Claiming that his health precluded teaching, Behring devoted himself to research and business ventures. In 1914, he founded the Behringwerke for the production of sera and vaccines. His career provides a paradigm for a new era in which studies of basic science could lead to patents and proﬁts.
During the nineteenth century, several particularly virulent out- breaks of a disease variously known as croup, malignant angina, and throat distemper attracted the attention of clinicians and bacteriologists. Pierre Fide`le Bretonneau (1778–1862) suggested the name ‘‘diphtheritis’’ for what he thought of as a speciﬁc form of malignant sore throat that killed young children by sudden suffocation. In 1883, Corynebacterium diphtheriae, the bacillus that causes the disease was discovered by Theo- dor Klebs (1834–1913) and Friedrich Loefﬂer (1852–1915). By the end of the decade, researchers at the Pasteur Institute in Paris had demon- strated that bacteria-free ﬁltrates of diphtheria cultures contained a toxin that produced the symptoms of the disease when injected into experi- mental animals. Autopsies revealed that the disease caused considerable damage to the internal organs, but the bacteria usually remained local-
ized in the throat. Pasteur’s associates E´ mile Roux (1853–1933) and
Alexandre Yersin (1863–1943) proved that diphtheria bacilli release tox- ins that enter the bloodstream and damage various tissues. Diphtheria is acquired by inhaling bacteria released when a patient or carrier coughs and sneezes. Within a week after infection, the victim experiences gener- alized illness and the characteristic ‘‘false-membrane’’ at the back of the
throat. During virulent outbreaks, the disease had a case fatality rate of
30 to 50 percent, but many people acquired immunity after experiencing fairly mild symptoms. Doctors sometimes performed tracheotomy to prevent death by asphyxiation, but even if this operation produced temporary relief, toxemia might still cause death. Tracheotomy was essentially replaced by intubation in the 1890s.
Shibasaburo Kitasato (1852–1931), a Japanese physician working at Koch’s Institute, isolated the tetanus bacillus and proved that, like the diphtheria bacillus, it produced a toxin that caused the symptoms of the disease when injected into experimental animals. Trained as a military doctor in the Listerian era, Behring was intrigued by the pos- sibility of using ‘‘internal disinfectants’’ against infectious diseases. Experiments with iodoform initiated a life-long preoccupation with antitoxic substances and an appreciation for the fact that chemical dis- infectants were often more damaging to the tissues of the host than to the invading bacteria. Some preliminary experiments indicated that while iodoform did not kill microbes, it seemed to neutralize bacterial toxins.
Working together on the toxins of diphtheria and tetanus bacilli,
Behring and Kitasato demonstrated that when experimental animals were given a series of injections of toxins, they produced antitoxins, sub- stances in the blood that neutralized the bacterial toxins. Antitoxins produced by experimental animals could be used to immunize other ani- mals, and could even cure infected animals. Encouraged by these early results, Behring predicted that his toxin–antitoxin preparations would lead to the eradication of diphtheria, which typically killed more than ﬁfty thousand children in Germany each year.
A ﬁrst step in the transformation of serum therapy from a labora-
tory curiosity into a therapeutic tool was accomplished by turning sheep and horses into antitoxin factories. Although Behring planned to enter a commercial relationship with Hoechst, the German chemical company producing Koch’s tuberculin, his preparations were too variable, unre- liable, and weak for routine use or commercial distribution. Fearing that French scientists would make further advances in serum therapy, Behring asked Paul Ehrlich (1854–1915) for help. Having systematically worked out methods of immunization with the plant toxins ricin and abrin, Ehrlich knew how to increase antitoxin strength and measure the activity of antisera with precision. By producing highly active, stan- dardized sera, Ehrlich made serum therapy practical. Behring and Ehrlich established a laboratory in Berlin to obtain serum from sheep and horses.
In 1892, Berhing, Ehrlich, and Hoechst entered into an arrange- ment to work on diphtheria antitoxin. Production and marketing of the therapeutic serum began two years later. According to their previous agreement, Behring and Ehrlich were to share in the proﬁts from
diphtheria antitoxin, but Behring persuaded Ehrlich to give up his share of the proﬁts by promising to help him get his own research institute. For reasons that remain obscure, Behring did not carry out his part of the deal. He did, however, keep his enlarged share of the proﬁts and became a very wealthy man. The immunity provided by Behring’s therapeutic serum, which was a result of passive immunity, was short- lived. In 1901, Behring began experiments with attenuated cultures of diphtheria bacilli as a means of establishing active immunization. In
1913, Behring publicly described his diphtheria protective agent, which was called ‘‘Toxin–Antitoxin.’’ It contained a mixture of diphtheria toxin and therapeutic serum antitoxin.
Relations between Ehrlich and Behring rapidly deteriorated as
Behring became richer and more arrogant. Perhaps Ehrlich could take some comfort in the fact that after their collaboration ended all of Behring’s scientiﬁc projects were failures. Koch’s tuberculin ﬁasco stim- ulated Behring’s search for an effective therapeutic agent, but he too was unsuccessful. Instead, he attempted to develop a preventive vacci- nation. Assuming that the tubercle bacillus was primarily transmitted to children through milk, Behring tried to destroy this source of infection by treating milk with formaldehyde. Even if babies or calves could be forced to consume formaldehyde-treated milk, most tuberculosis infections were contracted via the respiratory route. Behring’s attempts to establish attenuated tubercle bacteria that could serve as immunizing agents were unsuccessful.
Diphtheria was generally considered a minor disease when com- pared to tuberculosis, but while tuberculin was causing bitter disap- pointment, serum therapy was being hailed as a major contribution to medicine. The ﬁrst Nobel Prize for Physiology of Medicine, awarded in 1901, honored Behring for creating a ‘‘victorious weapon against ill- ness and deaths.’’ By making it possible to induce life-saving active and passive immunity, serum therapy seemed to be the ultimate answer to the threat of infectious diseases. Yet within 10 years, the euphoria trigged by the success of the diphtheria antitoxin was replaced by pro- found disappointment and the dawn of a period that has been called the ‘‘Dark Ages of Immunology.’’ Despite the overall success of anti- toxin, some patients experienced serious side effects and a few died. Treatment was most successful if given in the early stages of the disease, but doctors were reluctant to use antitoxin until the disease was clearly life threatening. Control programs were complicated by the discovery that many people were asymptomatic carriers.
By the end of the twentieth century, genetic engineers were exploit-
ing the ‘‘naturally engineered’’ properties of various bacterial toxins in order to create hybrid molecules in which toxins are linked to speciﬁc antibodies. Diphtheria toxin, for example, was naturally engineered as a protein that could penetrate cell membranes, but it is only one of several
bacterial toxins that have found a place in biomedical research and medical practice. The use of botulinum toxin for cosmetic purposes is, perhaps, one of the best-known examples. Previously, Clostridium botu- linum toxin was universally feared as the cause of paralysis following the ingestion of improperly preserved foods. Just as alchemists once began their quest for powerful elixirs with poisons, genetic engineers have turned to bacterial toxins to ﬁnd molecules suitable for appropriate modiﬁcations. Such novel immunotoxins have been referred to as ‘‘poi- soned arrows’’ or ‘‘smart bombs,’’ which, at least in theory, can deliver more ﬁre power than the ‘‘charmed bullets’’ ﬁrst synthesized in the laboratory of Paul Ehrlich, the founder of chemotherapy.
Since the discovery of serum therapy, diphtheria has been the most
successfully studied of the once common childhood diseases. Case fatal- ity rates rarely exceeded 10 percent, but, sometimes, exceptional epi- demics took a very heavy toll among young victims. Because immunity can be brought about by antibodies directed against the toxin itself, researchers could focus on the toxin rather than the bacillus. In 1928, Gaston Leon Ramon (1886–1963) discovered that diphtheria toxin treated with formaldehyde retained serological speciﬁcity and immuno- genicity, while losing its toxicity. Modiﬁed toxins were called ‘‘toxoids.’’ Evidence for the proposition that there is nothing new under the sun can be found in nineteenth-century reports about certain ‘‘wizards’’ in cen- tral Africa who told visiting Europeans that they could protect people against snakebites with a potion containing snake heads and ant eggs. Native healers in other parts of the world have employed similar meth- ods. By exploiting the fact that certain ants contain formic acid, so-called primitive healers had accomplished the chemical detoxiﬁcation of toxins and venoms. Massive immunization campaigns have almost eliminated the threat of diphtheria in the wealthy industrialized nations. Diphtheria remains the only major human infectious disease of bacterial origin that has been so successfully managed by preventive immuni- zations. Unfortunately, a generation unfamiliar with the threat once posed by diphtheria is unable to understand the dangers posed by the breakdown of ‘‘herd immunity.’’