SERUM THERAPY

12 May

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.’’

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