Meanwhile, the German team, consisting of Koch, Georg Gaffky, and Bernhard Fischer, performed many autopsies on patients killed by cholera and were eventually able to isolate the responsible organism.28 Already on the road, the team then proceeded to Calcutta, where additional cholera samples were isolated. By early January 1884, still in India, Koch announced the isolation of a new bacterium, Vibrio cholerae, with the odd shape “a little bent, like a comma.”29 Koch’s updates on his work in Egypt and India were being closely followed by the German press, which applauded his findings. Ironically, the conclusive evidence that the organism isolated by Koch was responsible for cholera failed Koch’s own postulates (for technical reasons having to do with how the organism propagates). Thus, while Koch was ultimately proved correct, international skepticism, perpetuated by the rival French team and by their English allies, reigned supreme until the preponderance of scientific thought eventually swung in favor of Koch.30
An investigator at the Pasteur Institute in Paris would follow up on Koch’s discovery of the cholera bacterium and utilize Pasteur’s attenuation techniques to develop a vaccine. Waldemar Mordecai Haffkine was born to a Jewish schoolmaster in Imperial Russia (modern Ukraine). He was trained by Elias Metchnikoff, a Russian immunologist who later shared the 1908 Nobel Prize with Paul Ehrlich.31 Following the assassination of Tsar Alexander II in 1881 and the resulting pogroms that hunted down many in the “Jewish intelligentsia,” Haffkine joined the resistance but was soon thereafter injured and jailed, released only after the venerable Metchnikoff vouched for him.32 In 1888, Haffkine emigrated to Switzerland and followed his mentor Metchnikoff to the Pasteur Institute in Paris. Neither scientist ever returned to Russia, and Haffkine found the only job he could in Paris: as a librarian in the Pasteur Institute.
Haffkine’s talents were soon liberated from the book stacks, and he initiated a campaign of chemical attenuation to develop a cholera vaccine.33 As seems to have happened far too many times in a more naïve age, Haffkine first tested his vaccine on himself on July 18, 1892. He survived this lethal experiment and reported on his findings a mere twelve days later in a scientific report to the English Linnaean Society.34 Still questioning whether Koch’s bacterium had been the organism responsible for cholera, neither Pasteur nor Metchnikoff was convinced of the finding. Unperturbed, Haffkine was convinced his new vaccine held promise and was determined to test his vaccine in people.
By the time of, and perhaps due to, word of Haffkine’s key study, the British biomedical community had begun to accept that Vibrio cholera was the organism responsible for the disease and that Haffkine’s discovery might indeed hold value. As further incentive, cholera was a particular problem in the Indian colonies, and the British were highly motivated to investigate potential vaccines. As often happens, chance played a major role in Haffkine’s future. The British ambassador to France, Lord Frederick Dufferin, happened to be a former viceroy of India and took particular interest in Haffkine’s work (despite the fact that most of the French scientific establishment was still dubious about Vibrio cholerae and its links to the disease).35
Arriving in England, Haffkine met Ernest Hanbury Hankin. You may recall that Hankin was the English doctor who would later advance medical science by discovering the presence of an unknown substance in the polluted waters of the Ganges. We now know this to be the first description of a bacteriophage. Haffkine and Hankin initiated a lifelong friendship, and Hankin later followed Haffkine to India, where they also collaborated on a series of scientific partnerships.
Consistent with his passion to alleviate the scourge of cholera, Hankin served as Haffkine’s fifth guinea pig to test the safety of the attenuated cholera vaccine (after Haffkine himself and three Russian exile friends). Hankin also helped Haffkine by communicating the encouraging findings to the prestigious British Medical Journal, which published his findings mere weeks before Haffkine set off to initiate a much more ambitious campaign of clinical trials in India.36
The trip was made possible because Lord Dufferin organized a meeting between Haffkine and Lord John Kimberley, the British secretary of state for India. Upon learning of the potential for a vaccine to prevent one of the most common diseases in the Raj, Kimberley embraced the idea and gave Haffkine access to all parts of the subcontinent. However, the British mandarin would only allow Haffkine to perform his studies under the condition that all subjects consent to participation in experiments (a relative rarity a half century before the development of the Nuremberg Code in the wake of Nazi atrocities during the Second World War). Haffkine was also forced to support his study with his own resources or donations. Undeterred, the adventurous Haffkine lobbied wealthy donors around the United Kingdom. Upon securing just enough funding to make the trip (but not for the clinical studies of the vaccine), Haffkine set out to confirm his belief in the cholera vaccine.
Haffkine arrived in Calcutta (now Kolkata) in March 1893. It was a monsoon-soaked metropolis that provided favorable conditions for the spread of a waterborne pathogen such as cholera. Unfortunately, the local authorities were not particularly enamored with the idea of a heavily accented Franco-Russian scientist conducting a series of dangerous medical experiments on their civilian population.37 Fortunately for Haffkine, Hankin had just returned to the subcontinent to resume his role as the head of the bacteriological laboratory at Agra in Uttar Pradesh. He asked his friend to conduct the cholera trials almost a thousand miles away, but this site was in the drier conditions of north central India (not particularly know for outbreaks of cholera). Nonetheless, Haffkine recognized the opportunity to leverage the support provided by Hankin’s offer and began the (voluntary) immunization of ten thousand British and Indian soldiers (at the government’s expense rather than his own).38, 39
Luck worked against Haffkine for a time, as the monsoon rains did not bring cholera to Uttar Pradesh that year.40 Although Haffkine was disappointed, a high-profile British official he’d met shortly after his arrival in India asked him to return to Calcutta and assist in evaluating an ongoing cholera outbreak. The Scottish-born Sir William John Ritchie Simpson was the lead public health officer for Calcutta and a pioneer in the emerging field of tropical medicine.41 In appreciation for Haffkine’s assistance in identifying the source of a cholera outbreak in his mandate, Simpson allowed Haffkine to initiate studies of his vaccine.
The human clinical trials began in earnest in the spring of 1894. Within months, the results of Haffkine’s Calcutta experiment were sufficiently impressive to expand the number of subjects and for Simpson to identify additional resources to underwrite the studies. Amidst this exciting progress, Haffkine was struck down by malaria and was forced to convalesce in Europe for a time. Consequently, the work was continued by Simpson in India while Haffkine convalesced in England. As word of success spread locally, Indian and colonial English subjects concerned about the spreading cholera outbreak volunteered en masse, and the number of subjects soon swelled beyond forty thousand.42
We will return to Haffkine later in this chapter, but it will be necessary to briefly summarize the rapid-fire development of vaccines, particularly in France during the final years of the 19th century and the early decades of the 20th.
Mad About Rabies
Pierre Victor Galtier was a veterinarian. Born in 1846 to poor parents, he was effectively orphaned by the age of seven.43 The young Pierre nonetheless demonstrated sufficient intellect to overcome a troubled start: he ran away from the nuns at the orphanage but became valedictorian upon his graduation from the Ecole Veterinaire de Lyon in 1873.44
After taking a position as a veterinarian and marrying the boss’s daughter, he joined his alma mater in Lyon in 1876, eventually rising to lead the departments of pathology and internal medicine.45 By 1879, Galtier published a thesis on rabies, which declared that the causative pathogen could be isolated from dogs and transmitted to rabbits. He also demonstrated that while changing species, the virus lost much of its vigor. What Galtier found (and which we now know
happens at the level of variability in the DNA molecule) was that the ability to productively infect a new species produced a range of variants—essentially mutants—that would be less well-adapted to causing disease in humans. Such understanding led Galtier to consider the potential to develop an attenuated pathogen as a vaccine. Many contemporaries did not believe rabies was caused by a virus; they presumed this to be a bacterium that had yet to be isolated or, perhaps, a bacterial toxin. Likewise, the response of German scientists was largely skeptical, as many adhered to the idea that the behavior and tenacity of microscopic organisms was fixed.
Despite these handicaps, Pierre was determined to identify a way to treat or prevent the disease. Their desperate need for such a treatment is illustrated by examples put forward in an English 1878 treatise on the subject written for physicians. A primary recommendation for treatment of rabies was a procedure known today as “cupping.”46 Specifically, a cup would be heated with cigar smoke and then placed over the wound. As the temperature cooled in the cup, a bit of suction was applied, which was intended to raise the virus to the surface of the wound. Then, a physician had a choice. He could either use a red-hot poker to scorch the wound, or, lacking an iron poker or fire, he could rely upon the ubiquitous presence of guns (back in the day when guns were more prevalent). In the latter case, a bullet was to be dismantled and then sprinkled into the wound. The doctor would then light a match and drop it into the wound. The resulting explosion might sometimes be sufficient to kill the infectious material, though the efficacy of this treatment was doubtful. In other words, rabies was predictably a death sentence.
To help avoid such desperate (and largely ineffective measures), Pierre published a manuscript in 1880 that provided an overview of what was known about the disease and its cause.47 This publication clearly inspired Louis Pasteur to take up work on a rabies vaccine, but the memory of Galtier and his key contributions have largely faded over the years. This oversight is perplexing, as Pierre continued his studies in animals, demonstrating in 1881 that the direct injection of the attenuated rabies virus into the blood could protect sheep from rabies. Despite a successful demonstration of the feasibility of a rabies vaccine, the relatively obscure country veterinarian was overshadowed by the dominance of the Parisian scientist.
Much as we saw with Toussaint, Pasteur took or was given credit for many ideas and outcomes that were more accurately attributable to Galtier. In 1882 Emile Roux, then an outstanding student in the Pasteur laboratory, began studies on rabies; his dissertation frequently cited the inspirations and background provided by Galtier.48 In contrast, Pasteur, in most of his published works and public talks, was dismissive of Galtier and his work. This lack of attribution literally pained the more junior investigator, who was sensitive to Pasteur’s lack of embrace and fearful of the possibility that Pasteur might emerge as an unfriendly rival.49 Although Roux would essentially copy Galtier’s studies and try to cite his predecessor’s work, Roux’s work was inappropriately hailed as the key breakthrough, particularly given Pasteur’s profile and advocacy for Roux.
In July 1885, Roux and Pasteur at last conducted a key study that Galtier had not. Despite lacking medical credentials, Pasteur tested his vaccine in a person. A nine-year-old boy by the name of Joseph Meister had been mauled by a rabid dog and was undoubtedly doomed to a slow, painful death from rabies (thankfully, he had not been subjected to the gunpowder approach). Master Meister’s only hope was the experimental vaccine being developed by Roux and Pasteur. The idea of treating the child was particularly dangerous, since Pasteur lacked a medical degree and could therefore be subject to legal prosecution.50 However, the child was otherwise doomed, and Pasteur agonized about whether to conduct such an ethically- and legally-fraught experiment. Prodded by his pediatrician colleague, Jacques-Joseph Grancher, Pasteur relented.
Starting two and a half days after the mauling, the child was injected a total of twelve times with a preparation of rabies virus isolated from the spinal column of infected rabbits (recalling that the virus lost much of its killing potential when transferred from a dog to a rabbit).51, 52 The viruses within the spinal cords had been further attenuated by drying the material for two weeks prior to its use as a vaccine. Meister survived. Eternally grateful to Pasteur, he served at the Pasteur Institute for the rest of his life.
As an aside, there’s an apocryphal story about Meister ultimately meeting his end at the hands of Nazi guards (or suicide in a different version) while protecting the tomb of Pasteur during the German occupation of Paris, but alas, this story is even more heartbreaking. Meister sent his family out of Paris as the German military columns approached, only to receive word that his family was killed during their escape. The distraught Meister then committed suicide by asphyxiation with a gas furnace. In a tragic plot twist, his body was found later that day, when his family safely returned to their apartment in Paris.53
Upon learning of Pasteur’s experience with the rabies vaccine and the ethical implications of using Meister as a human guinea pig, Koch was perhaps unsurprisingly dismissive of his rival’s success.54, 55 Upon reflection, Koch conceded the implications of the discovery and later instructed his Berlin team to utilize the same techniques to develop a vaccine that could be distributed in Germany. However, the acrimony felt by Koch towards Pasteur was still sufficient in 1893 that he begged out of attending a seventieth birthday celebration, citing personal reasons (in his defense, he did end his twenty-six-year marriage with his wife that year). Three years later, Pasteur would be dead and Koch’s feelings themselves attenuated over time. By 1904, Koch felt sufficiently comfortable to visit the Paris headquarters of the Pasteur Institute, where he was warmly received.56
Toxoids
According to the website ScienceHeroes.com, the French scientist Gaston Ramon is estimated to have saved the lives of sixty million people.57 To put this in perspective, such an outcome equates to roughly the entire population of the United Kingdom or the number of people who voted for Donald Trump in the 2016 election (though three million less than his rival). Moreover, sixty million people is more than ten times the number of people saved by the extraordinary contributions of Gertrude Elion, who, as we saw, developed a toolbox of new medicines that included some of the most frequently used cancer and HIV/AIDS drugs.58
Despite this impressive feat, the name Gaston Ramon remains almost entirely unknown to modern ears, perhaps in part because his achievements never earned him the notoriety of a Nobel Prize. However, this was not for lack of trying, as Ramon holds the dubious record of being nominated on 155 separate occasions for a Nobel Prize, more than any person before or since.59
Gaston Ramon was the son of a baker, born in 1886 in the small town of Bellechaume near Paris.60 In 1906, he enrolled in the local veterinary school, l’École Vétérinaire d’Alfort, located in the Parisian suburbs. It was one of four veterinary schools in France. Gaston’s choice of Alfort was well timed and placed, as its director, Henri Valee, had deep connections with the Pasteur Institute. These contacts would serve Ramon well and in turn help revitalize the Pasteur Institute following the death of its eponymous and larger-than-life leader.
The Pasteur Institute was a government-backed research organization officially launched in 1888 to honor its founder.61 The principals list at the new institute was a who’s who of prominent scientists we have met throughout our story: Emile Roux, Charles Chamberland, Elie Metchnikoff, Alexandre Yersin, Jacques-Joseph Grancher, and Emile Duclaux.62 With the death of the great man on September 28, 1895, the eldest of the group, Duclaux (who was a mere forty-seven, while all the others were in or barely out of their thirties), was elected director to replace Pasteur. Duclaux had a long history with Pasteur, accompanying him during his studies of silkworms during the Franco-Prussian War, then leading the internationally recognized Annales de l’Institute Pasteur—a periodical that remains a leading source of microbiology research—from its beginnings. During his tenure as the leader of the high-profile institu
te, Duclaux took up the cause of Alfred Dreyfus.
Dreyfus was a Jewish officer of the artillery. Based on dubious evidence, he was wrongfully convicted in 1895 of selling secrets of new artillery innovations to the despised Germans.63 Given the magnitude of the crime, Dreyfus was publically humiliated before being exiled to a life of imprisonment on the notorious and squalid Devil’s Island off the coast of French Guiana. Over the following months, evidence was unearthed by Lt. Colonel Georges Picquart revealing that the actual traitor was Major Ferdinand Walsin Esterhazy, but Picquart was silenced and assigned to a post in the southern deserts of Tunisia. As word of the cover-up leaked to the press, it became clear that Dreyfus’s conviction reflected widespread anti-Semitism in the French High Command, and Dreyfus’s cause was taken up by the famous author Emile Zola. Joined by many prominent citizens, including Emil Duclaux, these dissenters eventually gained a pardon for Dreyfus in 1899. He was fully exonerated in 1906.
After Duclaux’s death in 1904, the reigns of the Pasteur Institute were handed over to Emile Roux.64 Roux, you may recall from the previous chapter, had led Pasteur’s efforts to develop an antitoxin for diphtheria. Roux also led the team that developed Pasteur’s rabies vaccine and the expedition that sought to isolate cholera in Alexandria (until the death of Louis Thullier). In 1901, Emile Roux recruited Henri Vallee, who had just rejoined his alma mater veterinary school in Alfort (and would soon rise to lead the institution). Roux and Vallee shared research interests in tuberculosis and foot-and-mouth disease, both of which are problems for both livestock and humans.
In 1911, Vallee introduced Roux to one of his most promising students, a newly credentialed veterinarian by the name of Gaston Ramon.65 An impressed Roux hired Gaston and tasked him with oversight of the production of a growing number of different horse-derived antisera, including antibodies targeting diphtheria and tetanus. As the Great War became entrenched in the north and western portions of France, disease outbreaks inevitably followed. As its part of the war effort, the Pasteur Institute was tasked with meeting the ever-increasing demand for antisera. Beyond simply manufacturing more material, Ramon was challenged with identifying ways to preserve these vital medicines. Once they left the institute, the sera were highly susceptible to contamination from bacterial contaminants, which ironically feasted on the proteins in the antitoxins as a food source. Ramon’s solution was to package the antisera with small amounts of formalin, the active ingredient of embalming fluid. Gaston and other at the Pasteur Institute had routinely used formaldehyde to sterilize the glass tubes and flasks, and he considered that including small amounts of this chemical in tubes of antitoxin might likewise suppress the growth of contaminating organisms.66 This simple idea worked and allowed the institute to increase the storage and transport of the antitoxins they produced while preserving their efficacy.
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