Tag Archives: Paul Ehrlich

Elie Metchnikoff: The “Father of Innate Immunity”

The legend of Isaac Newton being struck on the head by a falling apple has long been enshrined in scientific lore. Likewise, there is the tale of Mendeleev suddenly grasping the relationship between the elements (i.e., discovering the Periodic Table) while struggling over how to organize them for a chemistry textbook he was writing. And, there is the myth of Kekule envisioning the benzene ring structure while dreaming of a snake grasping its own tail. Also, there are the fables of Ben Franklin and his Kite, Darwin and his finches, and Galileo dropping objects from the Leaning Tower of Pisa, among others.

Here we have the tale of Russian zoologist Elie Metchnikoff (1845-1916) who, in 1882, discovered leukocyte recruitment and phagocytosis as key elements in the body’s natural defenses. The mythical aspect of Metchnikoff’s discovery is that it allegedly happened while he was experimenting on starfish larvae. Metchnikoff was awarded a share the 2008 Nobel Prize in Physiology or Medicine for his discovery. German microbiologist Paul Ehrlich shared the 2008 award for his pioneering discoveries in humoral immunity.

Elie Metchnikoff
Elie Metchnikoff

We are fortunate to have Metchnikoff’s account of his 1882 epiphany, written in his own words shortly after he was awarded the Nobel Prize in 2008 (1).

“One day, as the whole family had gone to the circus to see some exceptional trained monkeys, while I had remained alone at my microscope and was following the life of motile cells in a transparent starfish larva, I was struck by a novel idea. I began to imagine that similar cells could serve the defense of an organism against dangerous intruders. Sensing that I was on to something highly interesting, I got so excited that I started pacing around, and even walked to the shore to gather my thoughts.

I hypothesized that if my presumption was correct, a thorn introduced into the body of a starfish larva, devoid of blood vessels and nervous system, would have to be rapidly encircled by the motile cells, similarly to what happens to a human finger with a splinter. No sooner said than done. In the shrubbery of our home, the same shrubbery where we had just a few days before assembled a ‘Christmas tree’ for the children on a mandarin bush, I picked up some rose thorns to introduce them right away under the skin of the superb starfish larva, as transparent as water. I was so excited I couldn’t fall asleep all night in trepidation of the result of my experiment, and the next morning, at a very early hour, I observed with immense joy that the experiment was a perfect success! This experiment formed the basis for the theory of phagocytosis, to whose elaboration I devoted the next 25 years of my life.

So, at a time when virtually nothing was known about the body’s natural defenses, Metchnikoff proposed that the mobile cells (later dubbed “phagocytes” or cell-eaters), which gathered around the thorns in the starfish larvae, were agents of healing. Moreover, he proposed that those cells are the first line of an organism’s defense against invading pathogens. Metchnikoff’s use of starfish larvae in his breakthrough experiment owed to his interest in marine invertebrates which, in turn, reflected his broad interest in natural history.

Metchnikoff’s passionate interest in science, natural history, and marine invertebrates developed early in his life. In 1870, when he was barely 25 years-old, he was appointed a professor of zoology and comparative anatomy at the University of Odessa; a position he resigned in 1882 because of limited research opportunities in Odessa, and because of political instability in the Ukraine after the assassination of Alexander II. Metchnikoff’s pioneering experiments that year were carried out at a private laboratory in Messina. [Later, during the Soviet Era, Odessa University was renamed Odessa I.I. Mechnikov National University, in Metchnikoff’s honor.]

In 1888 Louis Pasteur recruited Metchnikoff to the Pasteur Institute, where he would spend the remainder of his career. There, under the influence of Pasteur and Emile Roux (with whom he developed a close friendship), Metchnikoff turned his attention from simple organisms to experimental infectious disease and immunity.

By the late 1880s, Metchnikoff’s hypothesis that leukocyte recruitment and phagocytosis played a key role in host defense was garnering considerable attention. However, much of that attention was hostile, mainly because Paul Ehrlich, in Germany, was concurrently promoting the role of antisera in the body’s defenses.  The resulting feud between French scientists at the Pasteur Institute and Ehrlich’s colleagues in Germany was dubbed the “Immunity War.” [The “Immunity War” also may have reflected nationalistic feelings left over from the quite real Franco-Prussian war of July 1970 to May 1971.]

It was not until after Metchnikoff and Ehrlich shared their 1908 Nobel award that immunologists recognized that Metchnikoff’s phagocytes were a feature of “innate immunity,” while Ehrlich’s antibodies were a feature of “adaptive immunity.” Eventually both schools of thought would be integrated into our modern understanding of immunity. Metchnikoff would be recognized as the “Father of Innate Immunity,” while Ehrlich would be recognized as the pioneer of adaptive immunity (see the Aside). But, Metchnikoff’s early dispute with Ehrlich may be one reason why he avoided attending the 1908 Nobel Prize award ceremony. Metchnikoff presented a delayed Nobel lecture in Stockholm in 1909.

[Aside: Innate immunity is so named because it is present at birth and remains unchanged throughout life. It is the body’s first response to an invasive pathogen. Innate immunity is fast because it recognizes molecular patterns that are characteristic of broad classes of microorganisms; doing so via receptors that are encoded in the germ line. In contrast, the adaptive immune system is highly specific, recognizing determinants that are unique to each invader; doing so via receptors that are not encoded in the germ line. The adaptive immune system also has a memory. The price for the adaptive system’s specificity is that activation can take 1 week or longer. Innate immunity is the more primitive of these systems. It is present in primordial invertebrates, including insects, worms and mollusks. In contrast, adaptive immunity is seen only in vertebrates.]

How true to fact is the starfish-based tale of Metchnikoff’s discovery? A recent review by Siamon Gordon (Oxford professor of cellular pathology) suggests that Metchnikoff’s own personal account may not be entirely accurate (2).  For instance, a review of the early scientific literature shows that at the time of Metchnikoff’s discovery, phagocytosis had already been described by others.  Intriguingly, a description of phagocytosis appeared in the 1862 novel Fathers and Sons by Turgenev; an author admired by Metchnikoff. In Turgenev’s novel, “the description is given by a nihilist doctor, Yevgeny Bazarov, who, like Metchnikoff, used the microscope to make his own observations (2).”

Nonetheless, Gordon asserts that Metchnikoff indeed carried out the starfish experiments which led to the discovery. Moreover: “What distinguishes his (Metchnikoff’s) discovery from other early descriptions is that he followed up the initial observation with a program of striking experiments, which convinced him that this was a far-reaching process of general biological significance (2).” [Another review by Gordon summarized Metchnikoff’s many considerable contributions (3), some of which are noted below (see Note).]

The “myth” of Metchnikoff’s discovery, like all such myths, often convey a misimpression of the nature of scientific discovery, since they do not sufficiently acknowledge the intense efforts, sustained over considerable periods of time, which are generally necessary to produce major breakthroughs. But, these myths are fun and they do enhance the lay-public’s awareness of science.

Metchnikoff became somewhat of a public celebrity in his later years when he advocated eating yogurt to promote good health and long life (4). Apropos our larger story, Metchnikoff’s promotion of yogurt consumption was inspired by his interest in phagocytes. It was based on his beliefs that 1) the infirmities of old-age happen when phagocytes are transformed from defenders against infection into destroyers of healthy tissue by autotoxins (i.e., toxins that harm the organisms in which they are produced) derived from “putrefactive bacteria” residing in the colon, 2) that these degenerative changes could be prevented by inhibiting the colon’s putrefactive bacteria, and 3) that the host-friendly lactate-producing bacteria in yogurt would inhibit the putrefactive bacteria in the colon. [Metchnikoff regarded the colon as a “vestigial cesspool,” which does little more than provide a reservoir for putrefactive bacteria.]

Metchnikoff’s yogurt-eating regimen attracted numerous adherents for a time, but it eventually fell out of favor (indeed it even was satirized), since the premises on which it was based were never verified. Nonetheless, the medical community has recently been using Lactobacillus acidophilus to effectively treat several conditions, including pediatric antibiotic-associated diarrhea, acute infectious diarrhea, and persistent diarrhea in children. So, might Metchnikoff also be viewed as the “father” (or grandfather perhaps) of the current probiotics craze?

References:

1. Metchnikoff E: My stay in Messina (Memories of the  past, 1908); in Souvenirs, Editions en Languese Etrangeres. Moscow, 1959 (translated from the French by Claudine Neyen). (w.karger.com/doi/10.1159/000443331)

2. Gordon S. 2016. Elie Metchnikoff, the Man and the Myth. Journal of Innate Immunity, 8:223-227.

3. Gordon S. 2008. Elie Metchnikoff: Father of natural immunity. European Journal of Immunology, 38:3257-3264.

4. Mackowiak P. 2013. Recycling Metchnikoff: Probiotics, the Intestinal Microbiome and the Quest for Long Life. Frontiers in Public Health. 1-3.

Note: “His (Metchnikoff’s) notable observations include proof that organisms were taken up by an active process, involving living, and not just scavenged dead organisms; acidification of vacuoles, digestion and destruction of degradable particles including many infectious microbes including bacteria, spirochaetes and yeasts; uptake of host cells, e.g. erythrocytes, often nucleated for ready identification, from diverse species, as well as spermatocytes; and carmine dye-particles, used as an intravital marker of phagocytosis. Metchnikoff emphasized observations in living systems, combining microscopy and staining with neutral red and other histological labels to evaluate the acidity of vacuoles, viability and fate of ingested organisms. The bacteria examined included Cholera vibrio, Bacillus pyocyaneum, Bacillus anthracis and its spores, Mycobacterium (human, avian and bovine), plague bacilli, Streptococci and Gonococci, and some of these were studied in combination. He demonstrated killing by leukocytic enzymes (‘cytase’). Metchnikoff made important contributions to understanding the entire process of inflammatory recruitment, described at length in his lectures on comparative inflammation. He observed diapedesis through vessel walls, aggregation of leukocytes at sites of inflammation and their tendency to fuse, and he dissected the role of endothelial, epithelial and mesenchymal cells, as well as of lymphatic drainage and nervous elements in the classic hallmarks of inflammation (oedema, rubor, calor, dolor, loss of function) and repair. By using simple organisms, he discovered the central role of phagocytosis in diverse biologic models. This work led naturally to studies on the clearance and fate of organisms after experimental administration via a variety of routes, e.g. intravenous, intraperitoneal, subcutaneous and even the anterior chamber of the eye (3).”

 

Felix d’Herelle, the Discovery of Bacteriophages, and Phage Therapy

Viruses infect all classes of cellular organisms. Those that infect bacteria are called bacteriophages, or phages for short. Some phages are referred to as “lytic,” since they cause the infected bacterium to lyse. In fact, the bacteriolytic effect of these phages actually led to their discovery by Frederick W. Twort in 1915, in London. Felix d’Herelle, made the discovery independently in 1917, in Paris, after he too observed bacterial lysis.

Twort noted that his “bacteriolytic agent” exhibited two of the defining properties of viruses; the ability to pass through filters that block bacteria, and the requirement for cells (bacteria in that instance) in order to proliferate. Nevertheless, whereas Twort considered the possibility that his bacteriolytic agent might be a virus, he seemed to favor the notion that it was an enzyme secreted by the bacteria.

In contrast to Twort, d’Herelle was quite certain that the phenomenon he observed was due to a virus; one able to infect bacteria. Moreover, unlike Twort, d”Herelle ardently followed up his initial observations. d’Herelle also gave bacteriophages their name; which means “bacteria eaters,” although this is not what actually happens. See Aside 1.

Felix d'Herelle
Felix d’Herelle

[Aside 1: As has happened in other instances—e.g., the discovery of HIV (1)—priority for a scientific discovery can be a matter of bitter dispute. In the case of Twort and d’Herelle, contention over priority for the discovery of bacteriophages eventually waned, as many in the scientific community came to accept that their discoveries were independent. However, the observable effect of lytic phages was reported even earlier, in 1896, by British bacteriologist Ernest Hankin, who found that an agent which lyses Vibrio cholerae can pass through filters. Two years later, the Russian bacteriologist Gamaleya noted a similar occurrence in Bacillus subtilis. Yet neither of these researchers pursued the phenomenon, which lay dormant until resurrected by Twort in 1915.]

d’Herelle’s 1917 report of his discovery contains several notable observations (2). First, he stated that the bactericidal microbe cannot be seen under his microscope: “From the feces of several patients convalescing from infection with the dysentery bacillus, as well as from the urine of another patient, I have isolated an invisible microbe endowed with an antagonistic property against the bacillus of Shiga.”

Next, d’Herelle noted that the invisible microbe can be isolated from patients only when the Shiga bacillus is present as well: “In convalescent cases…the antagonistic microbe disappears very soon after the disappearance of the pathogenic bacillus…I have never found this antagonistic microbe in…normal subjects.”

d’Herelle then asserted that the antagonistic effect of the antibacterial microbe is seen only in association with the Shiga bacillus. That is, the agent is host-specific: “I have never obtained an activity against other microbes: typhus and paratyphoid bacilli, staphylococci, and so on.”

d’Herelle went on to proclaim that the agent is indeed a “living germ.” That is, it can proliferate. Moreover, its proliferation requires the presence of the Shiga bacillus: “The antagonistic microbe can never be cultivated in media in the absence of the dysentery bacillus…This indicates that the antagonistic dysentery microbe is an obligate bacteriophage.” Note that this is the first known use of the term “bacteriophage.”

d’Herelle concluded his 1917 paper with a comment on the generality of his findings: “It is probable that the phenomenon is not restricted to dysentery, but that it is of general significance, because I have been able to observe a similar situation, even though weaker, in two cases of paratyphoid fever.” See Aside 2.

[Aside 2: Bear in mind how little was known about viruses in 1917, other than that they pass through filters that block bacteria, and that they require cells to proliferate. Our last posting featured the crystallization of tobacco mosaic virus by Wendell Stanley (3). In Stanley’s 1946 Nobel lecture, he recounted that when he began that project in 1932, “the true nature of viruses was a complete mystery. It was not known whether they were inorganic, carbohydrate, hydrocarbon, lipid, protein or organismal in nature.”]

d’Herelle’s next goal, which he pursued with a passion, was to develop therapies against bacterial infections that would be based on using bacteriolytic phages. He achieved his first success in 1919, when he stopped a typhoid plague in chickens, by means of a phage isolated from a fecal sample of a typhoid-infected chicken.

Later in that same year, d’Herelle attempted phage therapy for the first time in a human; a 12-year-old boy suffering from dysentery at the Hôpital des Enfants-Malades in Paris. d’Herelle successfully treated his young patient with a phage isolated from a fecal sample of another dysentery patient. He ingested the phage preparation himself, to test its safety, before administering it to the boy. Soon afterwards, he cured three additional dysentery patients, using the same phage preparation.

Phage therapy was soon booming, with thousands more successes being claimed. Remarkably, these successes were achieved despite the fact that neither d’Herelle, nor anyone else at the time, knew the actual nature of bacteriophages. See Aside 2 above, and Aside 3.

[Aside 3: The 1925 Pulitzer Prize-winning novel, Arrowsmith, by Sinclair Lewis, describes fictional efforts to develop phage therapy. I highly recommend it, if for no other reason than to get a glimpse of what it was like to do medical research in that earlier time. Interestingly, the idea for Arrowsmith was suggested to author Sinclair Lewis by his friend Paul de Kruif, then a scientist at the Rockefeller Institute, and later a well-known science writer, perhaps best known for his 1926 book, The Microbe Hunters.]

Some of the successes claimed by d’Herelle occurred in 1920 in Indochina, where he used phages from plague-infected rats to treat human plague patients. In 1927, in India, under the sponsorship of the British colonial government, he stopped a cholera epidemic by means of two procedures. In the first, he administered phage preparations directly to individual recipients. In the second, he seeded the wells of cholera-stricken villages with the phages. In both instances the phages were isolated from Indian cholera victims, and in both instances it was claimed that the epidemic was ended within 48 hours, whereas the conventional intervention procedures of day were said to achieve the same result only after 26 days.

Yet despite the thousands of successes claimed by d’Herelle and his adherents, many in the scientific community questioned the efficacy of phage therapy. A major reason was that d’Herelle’s clinical trials did not meet accepted standards of experimental rigor. Most importantly, he did not include appropriate placebo controls in those studies. Perhaps he did not want to deprive any of his subjects of the benefit of the treatment that he so strongly believed in. Yet he also failed to include placebo controls in his earlier studies in chickens.

Nonetheless, in the pre-antibiotic era, there were few alternative interventions against bacterial infection, other than vaccination (where apt) and raising standards of public hygiene. Consequently, many large drug firms, including American companies Eli Lilly, Parke-Davis, Abbott, and Squibb, produced phages to treat a variety of bacterial pathogens. d’Herelle himself founded a commercial laboratory in Paris to produce therapeutic phage stocks. He returned the profits from his commercial venture into the support his phage research.

Notwithstanding the often spectacular successes that could reasonably be attributed to phage therapy, it was not a panacea. For instance, the outcome of phage therapy could be inconsistent, in part because companies were producing phage stocks at a time when these agents were far from understood. Also, phages are highly host-specific. Thus, effective phage therapy depended on accurately identifying the infecting pathogen.

Interest in phage therapy waned in the United States and in Western Europe in the late 1940s. The major reason was the sudden availability of penicillin and other antibiotics, which were easier to use than phages—they could be effective even when the identity of the etiologic agent was not known—and they were more reliable as well. During the Second World War, supplies of penicillin were limited, and priority was given to the military. But, when the war ended in 1945, and the general public became aware of the new “miracle drug,” demand for it was huge and phage therapy quickly fell by the wayside.

But while antibiotics became readily available in the United States and in Western Europe after World War II, this was not the case in Eastern Europe and in the former Soviet Union, which were isolated from Western advances in antibiotic production. Consequently, interest in phage therapy remained high in the East after the war. In fact, hundreds of scientific papers concerning phage therapy were being published in Eastern Europe and in the former Soviet Union. Many of these papers were from the Eliava Institute in Tbilisi, in the former Soviet Republic of Georgia (see Aside 4). The Eliava Institute produced phages against numerous bacterial pathogens, including staphylococci, Pseudomonas, Proteus, and Shigella.

Eliava Institute Bacteriophages
Eliava Institute Bacteriophages

[Aside 4: Georgian microbiologist, Giorgi Eliava, met d’Herelle, and acquired his enthusiasm for phage therapy, while carrying out research at the Pasteur Institute in Paris between 1918 and 1921. In 1923, after Eliava returned to Georgia, he established a laboratory in Tbilisi (renamed the Eliava Institute in 1988) for the explicit purpose of researching phage therapy. In1937 Eliava invited d’Herelle to join him in Tbilisi, where the two might then collaborate. d’Herelle accepted, and after spending several months in Tbilisi, he decided to stay permanently. But, Eliava was arrested by the Soviet secret police and executed as a “People’s Enemy.” One version of events has it that Eliava was executed because he was a rival of the Soviet secret police chief, Lavrenti Beria, for the affection of a woman. d’Herelle fled from Georgia for his own life.]

The Hirszfeld Institute of Immunology and Experimental Therapy, in Wroclaw, Poland, is another Eastern European organization actively engaged in phage therapy research. Work at the Institute has largely been motivated by “the emergence of bacterial strains, which are highly resistant to virtually all available antimicrobial agents.” That quoted phrase, as well as the following, is from the Institute’s web site: “Since 1980 the specific bacteriophages have been used in our Laboratory for the treatment of over 1500 patients with suppurative (i.e. pus producing) bacterial infections, in which a routine antibiotic therapy failed. The results obtained so far showed that phage therapy is safe and highly effective (the majority of patients were cured).” See Aside 5.

[Aside 5: The Hirszfeld Institute was founded by Ludwik Hirszfeld, perhaps best known as one of the co-discoverers of the inheritance of ABO blood type. Hirszfeld was Jewish by birth, but converted to Catholicism. Nevertheless, in 1939 the Nazis removed him from his scientific posts and banished him to the Warsaw Ghetto, where he organized underground anti-epidemic measures and vaccination campaigns against typhus, and also taught secret medical classes. In 1943, a day before he was to be sent from the Ghetto to an extermination camp, he and his wife escaped and found refuge in small Polish villages, using false names. Hirszfeld resumed his scientific career after the war. Incidentally, during the war, d’Herelle was kept under house arrest by the German occupiers of Vichy, France.]

Although enthusiasm for phage therapy has remained high in Eastern Europe and in the former Soviet Union, enthusiasm there did not forestall the declining interest in phage therapy in the West. The Eastern European studies were published in Russian and, as a result, were largely unavailable to scientists in the West. Moreover, those studies often did not meet Western standards of scientific rigor, as they often lacked proper controls and they did not adequately describe experimental procedures. [See reference 4 for a detailed review and assessment of the Eastern European phage therapy trials.]

The standing of phage therapy in the West was also undermined by d’Herelle himself, who hurt his cause by vigorously advocating his abiding belief that natural recovery from bacterial infection results from the bacteriolytic action of phages. He based this assertion on his early finding that he could isolate lytic phages only from those patients who were convalescing from a bacterial infection (see above and reference 2). He also believed that long-lasting immunity is the result of the phages present in the recovered patient.

d’Herelle’s assertions concerning recovery from infection and acquired immunity were at odds with key discoveries then being made by pioneering immunologists, such as Nobel laureates Elie Metchnikoff and Paul Ehrlich, who demonstrated the importance of leukocyte recruitment and phagocytosis in recovery from bacterial infection. But d’Herelle continued to assert that neither phagocytosis nor antibodies could produce bacteriolysis, which he thought was necessary to completely destroy the bacteria. The scientific community responded to d’Herelle’s heresy as though support for phage therapy implied lack of support for key developments in immunology, serology, and vaccines.

Yet despite the scientific and social reasons for the decline of phage therapy in the West, phage therapy may yet undergo a resurgence here because of the ever increasing threat of antibiotic-resistant bacteria. For instance, the European Commission is now funding a research project, called “Phagoburn,” which involves collaboration between France, Belgium, and Switzerland, with the goal of evaluating phage therapy for the treatment of burn wounds infected with Escherichia coli and Pseudomonas aeruginosa.

Patients in the Phagoburn study will receive phage preparations made by the French company, Pherecydes Pharma, which now has more than 1,000 bactericidal phages in its collection. And while researchers in the West are eagerly awaiting results of Phagoburn, some EU citizens, who are suffering with antibiotic-resistant infections, have travelled to Georgia, to receive phage therapy there.

In March, 2014, the U.S. National Institute of Allergy and Infectious Diseases included phage therapy among the seven approaches it was considering to counter antibiotic resistance. Yet it remains to be seen whether American drug companies will invest in phage therapy. They might be hesitant because of a 2013 U.S. Supreme Court ruling that prevents patenting natural genes; a ruling that might also apply to phages isolated from nature. Obtaining a patent might also be impeded by the fact that the principle of phage therapy is now nearly a century old. What’s more, after the research, development, and patenting phases, the company must still secure approval from the U.S. Food and Drug Administration to treat humans with a self-replicating agent that has the ability to evolve.

Before moving on, somewhat more might be said regarding the promise of phage therapy in the era of antibiotic resistance. First, as stated above, the specificity of a phage for a particular host bacterium can be a disadvantage since; as a result, effective phage therapy requires accurate identification of the infecting pathogen. However, the host-specificity of phage therapy nonetheless provides a significant advantage, relative to broad-spectrum antibiotics. Phage therapy selects for resistant bacteria only among the targeted bacteria, while antibiotics select for resistant mutants of many bacterial species; not just for resistant mutants of the targeted bacteria. And, if a particular bacterium should become resistant to a particular phage, it is very much easier, and less expensive, to produce a new generation of phage mutants able to attack the phage-resistant bacterium than it is to develop new antibiotics to attack multi-drug resistant bacteria. Furthermore, antibiotics target the normal body flora, as well as infecting pathogens. Thus, antibiotics, but not therapeutic phages, may affect the overall microbial balance in a patient, which in turn may lead to serious opportunistic infections.

Interestingly, a 1945 paper in The Lancet, by F. Himmelweit, who was working at St. Mary’s Hospital in London (where penicillin was first discovered), reported that the combination of antibiotics and phage therapy gave positive outcomes in clinical trials, while greatly reducing the occurrence of penicillin-resistant bacteria. Yet this finding seems to have been completely ignored in the West, despite the fact that penicillin-resistant bacteria were emerging as soon as penicillin was put into practice.

Irrespective of the future of phage therapy in the West, Felix d’Herelle’s legacy would seem to be secure. In addition to pioneering phage therapy, he also firmed up the discovery of bacteriophages, and he was also the first to develop the plaque assay method for titrating and purifying a phage (2). Taken together, these additional achievements provided the foundation for the origin of molecular biology (5).

Remarkably, d’Herelle had only a high school education and he was self-taught in science. Yet in 1928, in recognition of his development of phage therapy, he was offered a tenured professorship by Yale Medical School, which he accepted. Earlier, in 1924, he received an honorary medical degree from the University of Leiden, as well as the Leeuwenhoek medal from the Royal Netherlands Academy of Arts and Sciences. The latter award is notable, since it is granted only once every ten years to the scientist deemed to have made the most significant contribution to microbiology during the preceding decade. It was especially meaningful to d’Herelle, since his idol, Louis Pasteur, received the same award in 1895.

In the 1960s d’Hérelle’s name appeared on a Nobel Foundation list of scientists who were deemed worthy of the Nobel Prize, but who did not receive it for one reason or another. d’Herelle is believed to have been nominated eight times for the Nobel award. So it is also remarkable that when d’Herelle died in Paris in 1949, he was virtually a forgotten man.

References:

1. Who Discovered HIV?, Posted on the blog January 23, 2014.

2. D’Herelle, F. (1917) Sur un microbe invisible antagoniste des bacilles dysenteriques (An invisible microbe that is antagonistic to the dysentery bacillus). Comptes rendus Acad. Sciences 165:373-375. [The English translation, here and above, appears in Thomas Brock’s Milestones in Microbiology, Prentice-Hall, 1961.]

3. Wendell Stanley: First to Crystallize a Virus, Posted on the blog April 23, 2015.4.

4. Sulakvelidze, A., Z. Alavidze, and J. G. Morris, Jr. (2001) Bacteriophage Therapy. Antimicrob Agents Chemother. 45: 649–659.

5. Norkin, Leonard C. (2010) Virology: Molecular Biology and Pathogenesis. ASM Press, Washington, D.C. See Chapter 1.

Louis Pasteur: One Step Away from Discovering Viruses

Louis Pasteur (1822-1895) is the subject of our first posting of the New Year. Pasteur was history’s greatest microbiologist and, perhaps, its most famous medical scientist. Pasteur was also an early figure in the history of virology for his 1885 discovery of a rabies vaccine; only the second antiviral vaccine and the first attenuated one (see Aside 1). However, the main point of this tale is that Pasteur let pass an especially propitious opportunity to discover that the rabies agent is one of a previously unrecognized class of microbes; a class that is fundamentally different from the already known bacteria. Its members are submicroscopic and grow only inside of a living cell. Pasteur was just one step away from discovering viruses.

Louis Pasteur
Louis Pasteur

[Aside 1: Attenuation is the conversion of a pathogenic microbe into something that is less able to cause disease, yet is still able to induce immunity. Edward Jenner’s 1798 smallpox vaccine, the world’s first vaccine, as well as the first antiviral vaccine, was not based on the principle of attenuation. Instead, it contained live, unmodified cowpox virus. Although hardly understood in Jenner’s day, his smallpox vaccine worked because cowpox, which is not virulent in humans, is immunologically cross-reactive with smallpox. Thus, the relatively benign cowpox virus induced immunity against the related, deadly smallpox virus (1).]

The distinctive nature of viruses would first begin to be revealed in 1887 by a scientist of much less renown than Pasteur; the Russian microbiologist Dmitry Ivanovsky. The virus concept would be further advanced in 1898 by the accomplished Dutch botanist Martinus Beijerinck (2). In any case, to better appreciate how anomalous it was that Pasteur did not discover viruses, we review the greatness of his earlier achievements. After that, we consider the opportune circumstance that he let go by.

Pasteur was a chemist by background. Thus, his first major scientific discovery, at 26 years of age, was as a chemist. It was his 1847 discover of molecular asymmetry; that certain organic molecules exist in two alternative molecular structures, each of which is the mirror image of the other. Additionally, pairs of these asymmetric molecules are chemically indistinguishable from each other, and balanced mixtures of them rotate the plane of polarized light.

Pasteur’s discovery of molecular asymmetry was one of the great discoveries in chemistry. Yet his research would take on a momentous new focus when he began to investigate the chemistry of fermentations. This new course was inspired by the fact that while asymmetric molecules are not generated in the laboratory, they are found in the living world. And, since asymmetric molecules are found among fermentation products, Pasteur hypothesized that fermentation is a biological process, which he proceeded to demonstrate in 1857, basically by showing that fermentation products did not arise in nutrient broth if any microbes that might have been present were either killed by heating or removed by filtration. What’s more, he showed that specific fermentations are caused by specific microorganisms. Additionally, he discovered that fermentation is usually an anaerobic process that actually is impaired by oxygen; a phenomenon known as the “Pasteur effect.” And, he put forward the notion of aerobic versus anaerobic microbes.

Pasteur put his experience studying fermentations to practical use when he came to the rescue of the French wine industry, which was on the verge of collapse because of the wine becoming putrefied. Pasteur showed that the problem was due to bacterial contamination, and then showed that the putrefaction could be prevented by heating the wine to 50 to 60 °C for several minutes; a procedure we now refer to as pasteurization. Wines are seldom pasteurized today because it would kill the organisms responsible for the wines maturing. But, as we know, pasteurization is applied to many contemporary food products, especially milk. Pasteur also aided the beer industry by developing methods for the control of beer fermentation.

Pasteur’s study of fermentations led to an experiment of historic significance for biology in general. In the 1860s, the ancient notion that life can arise spontaneously from nonliving materials, such as mud or water, was still widely believed. The emerging awareness of microbes in the 1860s did not change this belief. Instead, it led to the idea that fermentations and putrefactions result from the spontaneous generation of microbes. In 1862, Pasteur unequivocally dispelled this belief by a simple yet elegant experiment in which he made use of a flask that had a long bending neck that prevented contaminants from reaching the body of the flask. If the broth in the flask was sterilized by boiling, and if the neck remained intact, then the broth remained sterile. But, if the neck of the flask was broken off after the boiling, then the broth became opaque from bacterial contamination.

Taken alone, Pasteur’s achievements that are enumerated above would have been sufficient to have ensured his lasting fame. Nevertheless, Pasteur’s greatest successes were yet to come. In 1867 he put forward the “germ theory of disease.” By this time, the existence of a variety of microorganisms, including bacteria, fungi, and protozoa, was already well established. Pasteur’s new proposal, that microorganisms might produce different kinds of diseases, was inspired by his earlier experimental findings that different microorganisms are associated with different kinds of fermentations, and by his 1865 finding that a microorganism was responsible for a disease in silkworms that was devastating the French silk industry.

After Pasteur proposed his germ theory of disease, Robert Koch (another giant in the history of medical microbiology) established that anthrax in cattle is caused by a specific bacterium, Bacillus anthracis. Koch had taken a sample from diseased cattle and then used his new method for isolating pure bacterial colonies on solid culture media to generate a pure culture of B. anthracis. Next, he inoculated healthy animals with a portion of the pure culture. The healthy animals then developed anthrax. Finally, he re-isolated B. anthracis from the inoculated animals. This sequence of isolation, infection, and re-isolation constitutes Koch’s famous postulates, which provide the experimental basis for establishing that a specific microorganism is responsible for a specific disease.

Even after Pasteur confirmed Koch’s anthrax findings in 1877, some members of the medical establishment still rejected the germ theory of disease, mainly because Pasteur was a chemist by background, rather than a physician. Nevertheless, Joseph Lister, an English surgeon, admired Pasteur’s work on fermentation and was impressed by Pasteur’s disproving of spontaneous generation. Based on Pasteur’s demonstration of the ubiquity of airborne microorganisms (another of his noteworthy achievements), Lister reasoned that infections of open wounds are due to microorganisms in the environment. The aseptic techniques that Lister then introduced were responsible for dramatically reducing infections during surgery.

The following is one of my favorite parts of this story. In 1879, Pasteur made his first important contribution to vaccinology, when he discovered, by accident, that he could attenuate the bacterium responsible for chicken cholera (now known to be a member of genus Pasteurella), and then use the attenuated microbe as a vaccine. It happened as follows. Pasteur instructed his assistant, Charles Chamberland, to experimentally inject chickens with the cholera bacterium so that he, Pasteur, might observe the course of the disease. Then, just before a summer holiday break, Pasteur directed Chamberland to inject the chickens with a fresh culture of the bacteria. Chamberland may have been preoccupied with thoughts of the upcoming holiday, because he forgot to inject the chickens before leaving. When he returned a month later, he carried out Pasteur’s instructions, except that he injected the chickens with the now aged bacteria. What happened next was most important. The chickens that were inoculated with the aged culture developed only a very mild form of the disease. After that, Pasteur had Chamberland inject those same chickens with freshly grown, presumably virulent bacteria. The chickens still did not develop disease.

It is not clear why Pasteur instructed Chamberland to inoculate the freshly grown culture into the chickens that earlier had received the aged culture. Perhaps it was an accident, or perhaps Pasteur saw an opportunity to carry out a possibly interesting experiment. (The chickens had survived a mild infection by the aged culture. Might they now be resistant to freshly grown virulent bacteria?) In any case, Pasteur repeated the entire sequence of inoculating the chickens with an aged culture and then challenging them with a fresh culture. The outcome was the same as before.

Pasteur correctly surmised that the aging process (actually, oxidation by exposure to air) had attenuated the bacteria. And, he learned by experimentation that the virulence of the cholera microbe could be reduced to any desired extent by controlling its exposure to air. Most importantly perhaps, he discovered that the attenuated bacteria could induce resistance to the virulent bacteria and, consequently, could be used as a vaccine. Pasteur’s chicken cholera vaccine was the first vaccine deliberately created in a laboratory. What’s more, it was the first attenuated vaccine. See Aside 2.

[Aside 2: During the years that Pasteur was carrying out his vaccine studies, nothing was known regarding the physiological basis of immunity, or the determinants of virulence, or of mutations, or the underlying mechanism of attenuation that changed a deadly microbe into a harmless one that still could induce immunity. Considering the intellectual milieu in which Pasteur carried out his investigations, it is all the more remarkable that he achieved so much. And while Pasteur’s interpretations for how his attenuated vaccines worked were far from accurate, they are still impressive for their plausibility. Initially, he thought that the attenuated organisms might simply compete with the virulent organisms for a limited availability of nutrients in the host. Later, he thought that the attenuated organisms might release a toxin that blocked growth of the virulent organisms. The notion, that the host might actually initiate its own defense, began to emerge in 1890 when Emil von Behring and Shibasaburo Kitasato discovered that a host factor neutralized the diphtheria toxin. Kitasato then put forward the theory of humoral immunity, proposing that a host serum factor could neutralize a foreign antigen. In 1891 Paul Ehrlich used the term “antibody” for the first time, in reference to those serum factors.]

This account of the cholera vaccine brings to mind Pasteur’s famous remark, “Chance only favors the prepared mind.” Yet in the context of our larger story, it is an ironic statement, considering that Pasteur later missed an auspicious opportunity to discover viruses. But, before getting to that, we briefly note Pasteur’s work on his anthrax vaccine.

In 1879 Pasteur began to develop an anthrax vaccine, which, like the cholera vaccine, would be based on his principle of attenuation. And, as in the case of the cholera vaccine, Pasteur attenuated the anthrax bacillus by exposing it to oxygen. [History records that Pasteur and his assistants developed a second approach to attenuate the anthrax bacillus, based on their discovery that when the bacilli are cultivated at 42 or 43 degrees centigrade, they do not develop the endospores that are necessary to cause a virulent infection.] In 1881 Pasteur carried out a dramatic public demonstration of the effectiveness of his air-oxidized anthrax vaccine in livestock, causing many doubters to accept the validity of his work. See Aside 3 and the end note.

[Aside 3: Currently, the only FDA-licensed anthrax vaccine for use in humans is BioThrax, produced by Emergent BioDefense Operations Lansing Inc. BioThrax is generated from an avirulent, nonencapsulated mutant of B. anthracis. It does not contain any living organisms. As suggested by the name of the manufacturer, BioThrax was produced mainly for the U.S. Department of Defense, for use in case B. Anthracis might be used as a biological weapon. Thus, BioThrax is not available to the public. People who are exposed to B. anthracis are now treated with antibiotics (e.g., ciprofloxacin and doxycycline).]

Pasteur turned his attention to rabies in1880, when the problem of rabid dogs in Paris was getting out of hand. Once again Pasteur sought to develop a vaccine, and once again he wanted to apply the principle of attenuation. But, early on, he found that he could not grow the rabies agent in pure culture. Thus, he was not able to isolate the rabies agent. Moreover, he would need to devise new procedures if he was to grow and attenuate it. His solution was to develop methods for cultivating the rabies agent in the spinal cords of live rabbits. His method for attenuation was then suggested by his assistant, Emile Roux, who had been studying survival of the rabies agent in pieces of rabbit spinal cord that he suspended inside a flask. Following Roux’s example, Pasteur attenuated the rabies agent by air-desiccating spinal cords taken from experimentally infected rabbits that earlier had died of rabies. Each successive day of desiccation resulted in greater attenuation of virulence, such that an extract from a spinal cord aged for 14 days could no longer transmit the disease. What’s more, those extracts could be used as inoculums that prevented rabies in dogs that later were challenged with the virulent microbe.

Pasteur, himself, took saliva samples from rabid dogs. In one such incident, he used a glass tube to suck up a few drops of deadly saliva from the mouth of a mad, squirming bulldog that was held down on a table by two assistants. The assistants wore heavy leather gloves.

Here is another of my favorite parts of this story. In 1885, nine-year-old Joseph Meister was bitten repeatedly by a rabid dog. Young Joseph’s desperate mother then brought her son to Pasteur, hoping that he might help Joseph. But, any attempt by Pasteur to treat the boy was sure to provoke controversy. Pasteur was not a medical doctor. Moreover, his rabies vaccine had never been successfully used in humans. Furthermore, attenuation and vaccination were still new and contentious concepts. For these reasons, Pasteur rejected many earlier requests for help from people in France, and from abroad as well. But, in Joseph’s case, Pasteur relented, convinced that the boy would die if he did not intercede.

Pasteur gave young Joseph a series of 13 injections, one each day, in which each successive injection contained a less-attenuated (stronger) virus. Pasteur dreaded inoculating Joseph with the last shot in the series; a one-day-old vaccine that was strong enough to kill a rabbit. Emile Roux wanted no part in this episode and, in fact, withdrew from the rabies study because of it. But, Joseph never developed rabies, and millions of people subsequently received Pasteur’s anti-rabies treatments. [Pasteur’s attenuated rabies vaccine may not have been entirely safe for humans. Modern rabies vaccines are generally killed virus vaccines, prepared by chemically inactivating tissue culture lysates.] See Asides 4 and 5.

[Aside 4: Post-infection rabies vaccination works and, indeed, is necessary because (for reasons that are still not entirely clear) the human immune response against a natural rabies infection is not able to prevent the virus from reaching the central nervous system, at which point the infection is invariably fatal. Importantly, the incubation period between the time of the bite and the appearance of disease can be more than several months, and is never less than two weeks. Consequently, there is a substantial window of opportunity for the vaccine to cause the virus to be inactivated at the site of the bite.]

[Aside 5: In 1888, Emile Roux, working at the Pasteur Institute (see below), would confirm the existence of the diphtheria toxin by showing that injecting animals with sterile filtrates of liquid cultures of Corynebacterium diphtheriae caused death with a pathology characteristic of actual diphtheria.]

Pasteur worked hard to isolate the rabies agent, but he wrongly presumed that he should be able to grow it in pure culture. Finally, in 1884, he conceded that he had not been able to isolate and cultivate the rabies agent in a laboratory media. So, might that failure alone have been sufficient to cause Pasteur to think of the rabies agent in new terms? Perhaps not, since, at the time, the inability to cultivate a microbial pathogen was assumed to be a laboratory failure, rather than a reason to hypothesize that that the agent was something other than a bacterium. [Even with the eventual awareness of the uniqueness of viruses, the inability of virologists to cultivate viruses outside of an animal would remain a mystery, as well as an obstacle, well into the early 1930s (3).]

Pasteur also got sidetracked while trying to isolate the rabies agent. In 1880 he injected a rabbit with the saliva of a child who died of rabies. He then examined the blood of the rabbit after it too succumbed to rabies. Using his microscope, Pasteur in fact saw a microbe in the rabbit’s blood, which he thought might be the rabies agent. However, he later found the same microbe in the saliva of normal children. Ironically, this microbe, which Pasteur at first thought might be the rabies agent, was actually Pneumococcus pneumoniae, a major bacterial pathogen that was correctly identified several years later by Albert Frankel. Thus, Pasteur missed the opportunity to identify a bacterial pathogen that is much more important in humans than rabies virus. Moreover, and importantly, Pasteur never did see the actual rabies agent under his microscope. Thus, he was aware that the rabies agent might be unusually small in comparison to the usual bacteria.

Here is another bit of irony. The item (apparatus?) that initially played the key role in distinguishing viruses from bacteria was invented in Pasteur’s laboratory. It was the unglazed terra cotta filter, conceived by Charles Chamberland, which he used to provide a good supply of sterile water for Pasteur’s lab. Chamberland allegedly developed these bacterium-proof filters while experimenting with a broken clay pipe that he bought from his tobacconist.

Bearing in mind that Pasteur was never able to grow the rabies agent in pure culture, and that he never saw the rabies agent under his microscope, might he have thought that it might be a submicroscopic infectious agent that is different from bacteria in some fundamental way? I have not come across any definitive answer to that question. But, I feel safe to say that it is unlikely that anyone other than Pasteur might have seriously considered that possibility. Regardless, Pasteur did not take the next logical step, which would have been to see if the rabies agent might pass through Chamberland’s filters. Had he done so, he could have isolated the rabies agent from the rabbit spinal cords, and he would have discovered “filterable viruses” (see below).

That crucial step was taken for the first time in 1887 by the Russian bacteriologist, Dmitry Ivanovsky, who used Chamberland filters in his investigations into the cause of tobacco mosaic disease. Ivanovsky could not propagate the tobacco mosaic agent (later known as the tobacco mosaic virus) in pure culture. However, because of his finding that the agent could actually pass through Chamberland’s filters, Ivanovsky is sometimes credited for discovering viruses. Yet Ivanovsky did not accept his own results. He still presumed that the disease was caused a bacterium, and he thought that the filters were defective or, instead, that the disease was due to a toxin produced by the bacterium.

In 1898, Martinus Beijerinck, unaware of Ivanovsky’s earlier work, also could not see or cultivate the tobacco mosaic agent. In addition, he too found that the agent passed through Chamberland filters. Beijerink expected, and perhaps even hoped that the filters would remove the agent from diseased plant extracts, so that he might prove it to be a bacterium. But despite his possible disappointment, Beijerinck went one major step further. He demonstrated that the filtered sap from a diseased plant did not lose its ability to cause disease after being diluted by repeated passage through new healthy plants. Consequently, the filterable agent was replicating in the plant tissue and, thus, could not be a toxin.

Little is recorded about Ivanovsky, aside from his four-page report on the tobacco mosaic disease (see Aside 6). In contrast, Beijerinck was a major scientist, who made numerous important contributions, including the discovery of nitrogen-fixing bacteria and bacterial sulfate reduction (4). Yet even Beijerinck found it difficult to conceive that the filterable, incredibly small, submicroscopic tobacco mosaic agent might be particulate in nature. Instead, he famously described it as a “contagious living fluid.” Nonetheless, Beijerinck, a botanist by background, is often considered to be the first virologist.

[Aside 6: Ivanovsky’s four-page paper would be unremarkable if it were not for the single sentence, “Yet I have found that the sap of leaves attacked by the mosaic disease retains its infectious qualities even after filtration through Chamberland filters.”]

Pasteur was probably unaware of Ivanovsky’s findings, and he did not live long enough to know of Beijerinck’s. So, we do not know what he might have made of their results. Regardless, Pasteur remained one step away from making these discoveries himself.

In 1898, after the announcement of Beijerinck’s findings, Friederich Loeffler and Paul Frosch isolated the foot and mouth disease virus; the first virus isolated from animals. Next, in 1901, in Cuba, U.S. Army doctor Walter Reed isolated yellow fever virus (5); the first pathogenic virus of humans to be isolated. In 1903, Paul Remlinger, working at the Constantinople Imperial Bacteriology Institute, filtered and then isolated rabies virus. Despite these early achievements, it was not until 1938 that the development of the electron microscope made it possible to resolve that viruses are indeed particulate, rather than liquid in nature. See Aside 7.

[Aside 7: The term “virus” indeed appears in the scientific literature of Pasteur’s day. However, at that time “virus” referred to any microbe that might cause disease when inoculated into a susceptible human or animal. By the 1890s, the term “filterable virus” came into use, meaning an infectious agent which, like the tobacco mosaic virus, passed through filters that retained bacteria. But, bearing in mind that there was not even a consensus regarding the identity of the genetic material until the early 1950s, there would be no clear understanding of viruses until then. In fact, the classic, early 1950s blender experiment of Alfred Hershey and Martha Chase, which featured bacteriophage T4, played a key role in establishing DNA as the genetic material, while also elucidating the essentials of virus replication (2).]

In 1887 Louis Pasteur founded the Institute in Paris that bears his name. A minor irony is that the Pasteur Institute was founded as a rabies vaccine center. The Institute has since been the site of numerous major discoveries in infectious diseases. But we underscore here that it was the site where, in 1910, Constantin Levaditi and Karl Landsteiner demonstrated that poliomyelitis is caused by a filterable virus, and where Félix d’Herelle in 1917 discovered bacteriophages. And it was also the site where, in 1983, Luc Montagnier and Françoise Barré-Sinoussi were the first to isolate HIV (6).

In a fitting end to our story, when Joseph Meister grew up, he became the gatekeeper of the Pasteur Institute. Meister was still minding the gate at age sixty four when, in 1940, the Nazis invaded Paris. Legend has it that when Nazi soldiers arrived at the Institute and ordered Meister to open Pasteur’s crypt, rather than surrendering Pasteur’s resting place to the Nazis, Meister shot himself (7).

Pasteur Institute: Museum and Crypt
Pasteur Institute: Museum and Crypt

End note:

Science historian, Gerald L. Geison, wrote a controversial revisionist account of Pasteur’s achievements, that was based on Geison’s reading of Pasteur’s laboratory notes (8). Geison undermines Pasteur’s integrity and discredits some of his major accomplishments. For example, Geison asserts that Pasteur surreptitiously used the oxidation procedure of French veterinary surgeon, Henry Toussaint, when preparing his own widely acclaimed anthrax vaccine for its public demonstration.

Max Perutz, who shared the 1962 Nobel Prize for Chemistry with John Kendrew for their studies of the structures of hemoglobin and myoglobin, reviewed Geison’s book for The New York Review of Books (December 21, 1995). Perutz’s review, entitled The Pioneer Defended, contains a vigorous rebuttal of Geison’s claims. Geison responded to Perutz’s review in the April 4, 1996 issue of The New York Review of Books. Perutz’s counter-response immediately follows.

I make note of all this because Geison’s uncertain assertions are reported as unqualified fact in some accounts of Pasteur’s work. And, while Perutz’s representations are not entirely accurate, the review, the response, and the counter-response make a very interesting read.

References:

(1) Edward Jenner and the Smallpox Vaccine, Posted on the blog September 16, 2014.

(2) Norkin, L. C. Virology: Molecular Biology and Pathognesis, ASM Press, 2010. Chapters 1 and 2 review key developments towards the understanding of viruses.

(3) Ernest Goodpasture and the Egg in the Flu Vaccine, Posted on the blog November 26, 2014.

(4) Chun, K.-T., and D. H. Ferris,  Martinus Willem Beijerinck (1851-1931) Pioneer of general microbiology, ASM News 62, 539-543, 1996.

(5) The Struggle against Yellow Fever: Featuring Walter Reed and Max Theiler, Posted on the blog May 13, 2014.

(6) Who Discovered HIV?, Posted on the blog January 23, 2014.

(7) Dufour, H. D., and S. B. Carroll, (2013), History: Great myths die hard, Nature 502, 32–33. This note contains an update on the myth.

(8) Geisen, G. L., The Private Science of Louis Pasteur, Princeton University Press, 1996.