Between Hope and Fear

by Michael Kinch

  After receiving training from Escherich, Pirquet graduated in 1900 and started a stint as a military surgeon. He later returned to his hometown of Vienna, taking a job at the Children’s Clinic. In 1902, Pirquet began mentoring a promising new Hungarian medical student by the name of Bela Schick. This event was life-changing for both, as it triggered a lifelong friendship and professional partnership between the two (who were only separated by three years of age) that would greatly advance our understanding of the immune system. Pirquet and Schick’s early collaboration centered on the fact that a sizeable fraction of children treated with diphtheria antiserum tended to display a constellation of symptoms one to three weeks after receiving their first injection.61 These children often demonstrated fever, malaise, hives, itching, and severe joint pain, which was frequently accompanied by swelling of lymph nodes throughout the body. By comparing these clinical symptoms with the outcomes from basic research studies with animals, the two investigators realized that animal-derived sera were triggering an exaggerated immune response. Worse still, the response was amplified following each exposure to an antiserum (any antiserum), potentially culminating in death. A prominent example of this “serum sickness” reverberated at the core of the Germanic medical community when the two-year-old son of a prominent researcher, Paul Langerhans (who had earlier identified the key component of the pancreas that would later be shown to produce insulin), died within minutes of receiving diphtheria antitoxin.62

  Over time, Schick and Pirquet advanced understanding of this disorder, realizing the human immune system (and that of other mammals) often perceives and overreacts during its assault upon molecules perceived as foreign. This defense mechanism evolved in part because viruses have the annoying ability to pick up portions of their host DNA and incorporate it into their genes. This process can function to provide a type of shield meant to confuse the immune system. Therefore, the immune system developed a countermeasure to recognize material from foreign species (e.g., cows and horses) and even from other people (the reason for the rejection of transplants). As we have seen, these reactions themselves can be responsible for many diseases. As the process was elucidated, Schick and Pirquet realized that these exaggerated defensive measures contributed to diseases beyond serum sickness and included more mundane (but still troubling) issues such as responses to animal and plant productions. The team was the first to term these diseases as “allergies” and appreciated that a variety of different stimuli such as seasonal pollens, foods, insect poisons, or even man-made chemicals could likewise trigger responses akin to that observed with serum sickness.

  The recognition of the causes and effects of serum sickness motivated a series of efforts to minimize the potential for damage. Early ideas included purifying the efficacious horse (or other species) proteins away from all other horse proteins within the antitoxins. This concept led investigators to evaluate the proteins in serum that were responsible for the beneficial effects. We now know these to be large protein structures known as “antibodies,” but this had yet to be discovered during the lifetimes of Schick and Pirquet.

  Almost a half century later, within months after the Germans invaded Poland, sparking the Second World War, the American professor Edwin Joseph Cohn began to research ways to treat shock on the battlefield.63 As a slight diversion, we will briefly introduce the concept of shock due to its key role in our story. More accurately known as circulatory shock, this malady is a physiological response that occurs when the volume of blood (or, more accurately, the presence of blood proteins in the body) drops as a result of bleeding. The body responds with a vigorous drop in blood pressure and heart rate, which causes rapid degradation of vital organs such as the kidney, invariably leading to death unless rapid and dramatic intervention, such as blood replacement, begins.

  One treatment for circulatory shock is intravenous replenishment of blood. However, such resources may be precious on the battlefield or forward aid stations. Cohn knew that reconstitution of volume alone (e.g., with a saline-based solution) was insufficient to counter shock, as fluids alone do not contain the vital proteins sensed by the body. In analyzing the different components of blood, Cohn isolated these important proteins from blood using ethanol (alcohol), then separated them into different classes of proteins utilizing a laboratory-based machine he had invented. Cohn was quite proud of this accomplishment. During his scientific talks, he would often take some of his own blood and place it in the machine at the beginning of the talk. He would then conclude his presentation (about an hour later) by showing the audience the fractionated products that the machine had separated in the meantime. This approach unexpectedly created a spectacle during a 1951 seminar at the Instituto Tecnico in Lisbon, when the blood clogged a key valve and the piping burst, bathing the audience in his blood.64

  Cohn’s process of physical separation (known as “fractionation”) revealed five different groupings of proteins. Many of these went on to provide essential treatments used during the war and thereafter. For example, the proteins of the fifth fraction largely consisted of albumin, a prominent serum protein that could be mixed with fluids and injected into soldiers to prevent shock. Other fractions contained fibrin, thrombin, and other proteins involved in blood coagulation. Later, these purified proteins were used to halt bleeding, both on the battlefield and for the rare individuals who suffer from clotting disorders such as hemophilia. Of particular interest to our story are a series of proteins found in the second and third fractions. These molecules, collectively known as gamma globulins, comprise what we today refer to as antibodies.

  The modern field of American academic medicine has from its beginnings been heavily influenced by a prominent scientist with the surname of Janeway. The Janeways first came to New England in the 16th century after fleeing from religious persecution in France and soon spread throughout the English colonies.65 Reverend Jacob Jones Janeway was vice president of Rutgers College in New Brunswick, and his son, George Jacob Janeway, was a prominent New Jersey physician. George’s son, Edward Gamaliel Janeway, served as the health commissioner of New York in the late 19th century, while his son, Theodore Caldwell Janeway, was the first full professor at an innovative new medical school in Baltimore by the name of Johns Hopkins. Theodore’s grandson, Charles Janeway (Jr.), was one of the world’s most brilliant and creative immunologists in the heyday of the field during the third quarter of the 20th century.66 In between Charles Jr. and Theodore was Charles Sr., who also served as a founder of modern immunology.

  Charles Janeway Sr. started his research career in the months after Edwin Cohn demonstrated the feasibility of separating blood into its various protein components. His work with serum sickness confirmed that the use of horse serum–derived albumin was not feasible, in part due to serum sickness. However, his work soon turned to utilizing the same techniques on human donor blood in early 1941. Charles’s work helped optimize the amounts and types of blood products that could impede shock, and the albumin-based products rode along in the Allied landing craft during D-Day three years later.

  In parallel with his studies on albumin, Janeway was evaluating the products of Fractions II and III and isolated the aforementioned “gamma globulins.”67 Working with a medical intern by the name of Fred Rosen, Janeway demonstrated that these gamma globulins could help protect children who otherwise lacked a stable immune system from infection. The benefactors of this finding included premature infants and children suffering from chronic infections, both of whom have underdeveloped or insufficient host defense mechanisms. Over time, these gamma globulins were understood to be the same protective antibodies that Kitasato and Behring had generated many years before.

  Having discovered these “antibodies,” the question now turned to how and where these interesting molecules were produced in the body. Throughout this book we return to Pasteur’s quote “Chance favors the prepared mind” even though it has become a bit of a cliché. A prominent example is seen with the question of how antibodies are pro
duced in the body. In the mid-1950s, an Ohio State University poultry scientist by the name of Bruce Glick was studying an organ near the anus of the chicken known for years as the bursa of Fabricius. The organ had been first described by Fabricius ab Aquapendente, a 17th-century Italian anatomist. However, the function of this small bud of tissues had remained unknown.68 Glick noted the bursa grew rapidly in the first three weeks after hatching but atrophied thereafter, much as we have seen with the thymus. To get at the function of this anatomical site, Glick surgically removed the bursa from adult birds but did not note any particularly interesting effects.69 Months later, a graduate student by the name of Timothy Chang asked Glick for some chickens to prepare antibodies against Salmonella bacteria. All that Glick could provide were some older chickens, whose bursas had been removed months before.

  Despite multiple attempts, Chang could not produce antibodies in those chickens lacking a bursa.70 Moreover, the animals that he injected often died of Salmonella even though he was using a weakened strain that should not have killed the chickens. Follow-on studies by Chang and Glick demonstrated that the bursa plays a key role in nurturing the cells that produce antibodies. Moreover, much as had been seen with the thymus, the bursa of Fabricius was only needed for this function in the first weeks of life. Removal of the bursa in an adult chicken did not impair its ability to make antibodies, since the job of the bursa had already been completed. Given the origin of these cells in the bursa, the antibody-producing cells have thereafter been known as B cells. (The human equivalent of the bursa of Fabricius is not itself a distinct organ but is found in the bone marrow.)

  Over time, concerns about serum sickness and other immunological responses against the horse-produced components of many antitoxins and antisera drove new research for alternative medicines. Whenever possible, human sources of B cell–derived antisera (gamma globulins) were procured. A prominent example is intravenous immunoglobulins (IVIG), which provides a source of immunoglobulins from healthy individuals that protect people with immune system deficiencies (such as those receiving a bone marrow transplant or following certain chemotherapies). More focused products included antibodies derived from people who had been exposed to (and successfully fought off) infectious diseases caused by cytomegalovirus (CMV) or respiratory syncytial virus (RSV). However, the breadth of isolating such medications is necessarily limited by the ability to find appropriate donors. In considering the much-needed antibodies to neutralize bacterial toxins, it is obviously unethical and impractical to generate human antisera against powerful poisons such as botulinum toxin or various snake and spider venoms. Many of these are therefore still generated in nonhuman species (such as horses).

  A key feature that distinguishes these first-in-class antisera from a new generation that would follow is that these animal- or human-derived sera are polyclonal in nature. This term refers to the fact that a wide variety of different antibodies is elicited during a typical immune response, and the complexity increases even further when one considers that any given batch of product likely contains antibodies from hundreds, or even thousands, of different donors. Thus, these sera may target a wide variety of different sites on a “foreign” molecule. A strength of this approach, as appreciated by nature, is that the larger the number of sites that is targeted (known as epitopes), the more likely that a particular antibody binding site will be useful in targeting a perceived foreigner. The disadvantage is that the number of antibodies that can target any given epitope will necessarily be diluted by all the other antibodies targeting other epitopes. Even a particularly useful antibody will also be subject to some degree of dilution. Since not all the antibodies produced are equally efficacious, much effort was placed into emphasizing only the most potent antibodies. Even more ideally, it would have been useful to identify a single antibody that works better than all the rest and then produce only this most superior molecule. However, such pipe dreams remained as such until the comparatively recent revelation of a powerful new technology.

  Biotechnology advances in the latter half of the 20th century utterly changed the prospects of making highly selective and safe antibody-based therapeutics. In the early 1970s, two scientists from the University of Cambridge (United Kingdom) developed a technique that would allow investigators to immunize animals and then isolate individual antibodies of interest. Georges Köhler and Cesar Milstein developed an elegant approach to fuse normal B lymphocytes and cancer cells using a derivative of the antifreeze routinely used to prevent overheating in automobile radiators. The resulting cells recall an ancient Greek mythological creature known as a chimera (a hybrid animal composed of parts from other animals such as the Sphinx), and these fused cells were known as hybridomas. The advantage of a hybridoma is that a single cell (known as a clone) can produce a single antibody that is not subject to the mutation and gene rearrangements that can alter an antibody in normal B cells. Unlike conventional immune cells, these hybridomas could also continue to grow indefinitely (reflecting the fact they are partially derived from cancer cells).71 As such, these hybridomas could produce a single antibody that recognized a single epitope. This breakthrough is today known as a monoclonal antibody (meaning all the antibodies are derived from a single clone). Beyond providing a more consistent product that does not vary from donor to donor, this technology yields other advantages: genes from these monoclonal antibodies can be isolated and engineered to modify their behavior, including improvements in how well they function and, when necessary, ways to improve their safety, ability to be administered in the body, or even the efficiency by which they can be manufactured.

  Over the past three decades, the field of monoclonal antibody research has evolved quickly. Among the most important contributions, investigators such as Greg Winter (also of Cambridge University) demonstrated that antibodies isolated from mice could be subject to genetic manipulation such that most of their mouse-derived structure is replaced by human sequences. In doing so, these products look to the immune system as if they are human and thus are not subject to rejection. Neither do they trigger adverse reactions such as serum sickness.72 A variation on this theme was the creation of laboratory mice that possessed human immunoglobulin genes and therefore also could avoid rejection.73 Greg Winter also reenters the story with improvements that arose from the use of genetically-modified bacteriophage.74 In this case, he cleverly inserted certain portions of immunoglobulin genes into bacteriophage, which could be used to infect E. coli bacteria in order to facilitate the creation of antibodies within hours (rather than the months that are otherwise needed to endure the immunization and isolation of antibodies from mice). Such biotechnology advances in monoclonal antibodies have revolutionized many fields of medicine, especially oncology, and have begun to do the same in our wars against infectious diseases. For now, we will conclude the chapter with a brief return to Schick and Pirquet to assess the work they did following their initial descriptions of serum sickness.

  Diagnosis and Disease

  Eclipsing even the identification and causation of serum sickness, Schick and Pirquet’s biggest contribution was the recognition that the same basic causes that lead to allergic responses could be used to predict whether an individual had been exposed to tuberculosis or other diseases. Tuberculosis has long plagued man and can insidiously remain dormant for long periods of time, not revealing who is infected until the symptoms have progressed to a point where intervention is challenging, if not impossible. Schick and Pirquet developed a skin test to assess whether an individual may be harboring tuberculosis. This test was optimized by the French researcher Charles Mantoux in 1908.75

  Separately, Schick and Pirquet emigrated to the United States. Pirquet accepted a position at Johns Hopkins in 1909 but suddenly changed his mind and abandoned the position to sail back to Europe within a year.76, 77 This timing was fortuitous, as the Austrian had returned just in time to take over as head of the Vienna Children’s Clinic after Escherich unexpectedly died in 1911. However, in another sense, t
his rather rash decision was a sad prelude to a spate of erratic behavior by Pirquet that worsened with age. Over the next few years, Pirquet became delusional and violent, as evidenced by his sudden decision to leap towards and vault out of a second-story conference room in the middle of a business meeting. His personal life was also troubled by a marriage that left him estranged from his wealthy relatives, causing further stresses, both personal and financial. Worse still, his wife began displaying her own signs of mental distress. Tragically, the bodies of Pirquet and his wife were discovered in their home on February 28, 1929, the victims of a double suicide.

  The story with Schick is far more cheering. The native Hungarian emigrated to the United States in 1923 and critically contributed to the public health of his adopted country by spearheading campaigns to eliminate diphtheria. Using the same approach he and Pirquet (and Mantoux) had earlier used to develop the tuberculin test, Schick developed a test to assess exposure to diphtheria. The test was widely deployed to help eradicate the disease.78 Whereas 100,000 Americans suffered from diphtheria in 1927, the disease was virtually eliminated within a few years. This grand outcome was in no small part due to Schick’s contributions, both in terms of his test and in leading an advertising campaign (in partnership with the Metropolitan Life Insurance Company) that distributed more than 85 million pieces of literature advising parents about immunization and other ways to keep their children safe from diphtheria.79

  With this history in mind, we now turn our sights to active immunotherapy, or, as they are better known, vaccines.