Prepared minds and the introduction of imaginon for hepatitic contagions
Authors: Adrian Reuben
Journal: Hepatology (2005)

Dans les champs de l'observation le hasard ne favorise que les esprits préparés. "In the fields of observation," observed Louis Pasteur in a lecture at the University of Lille on December 7, 1854, "chance favors only the prepared mind." Nineteen fifty-seven was an eventful year. The Soviets launched Sputnik and also tested their first intercontinental ballistic missile. Anthony Eden resigned the British premiership, having committed in the previous year what was then sarcastically referred to as political "Suizide" by colluding with the French and embroiling his country in the Israeli-Egyptian war. Althea Gibson and Lew Hoad won individual titles at Wimbledon, and Michael Todd's "Around the World in 80 Days" won an Academy Award for the best motion picture. Albert Camus was that year's Nobel laureate in literature, Leonard Bernstein's "West Side Story" brought musical violence to the Broadway stage, and Humphrey Bogart, Arturo Toscanini, and the ignoble un-American Senator Joseph McCarthy died. Nobel prizes in Chemistry, Physics, and Physiology or Medicine were given to scientists from the United Kingdom, China, and Italy, respectively, and Alick Isaacs and Jean Lindenmann (Fig. 1) published their discovery of interferon1, 2 from the lush leafiness and rolling fields of the northwest outskirts of London. (Left) Alick Isaacs (1921-1967) and (right) Jean Lindenmann (b. 1924) photographed during halcyon days at The National Institute for Medical Research. Photographs and permission to reproduce them generously provided by the office of Sir John Skehel, Ph.D., F.R.S., F.Med.Sci., Director, and Head of Infections and Immunity Group, MRC National Institute for Medical Research, Mill Hill, London, UK. The notion that viruses commandeer cells and subversively persuade them not to accept other viral guests, a phenomenon termed "viral interference" by Gerald Findlay and Frank MacCallum,3 had been entertained since the 1930s.3-5 The actual history of viral interference is certainly far older, however. Edward Jenner observed an attenuated response to vaccination in a boy who "sickened with the measles" the day before the discoverer of immunization inoculated him with "cow-pox matter."6 Jenner was almost certainly witnessing an interferon-mediated effect when he saw "…a deviation from the ordinary habits of the smallpox, as it has been observed that the presence of the measles suspends the action of variolous matter."6 In the 1940s, Werne Henle and his wife Gertrude Szpinglier Henle were leading investigators in the field of viral interference. Aside from their early7 and continuing work8 on interference, the Henles were most well known for their research on Epstein-Barr virus, including its causal relationship with infectious mononucleosis and two human cancers, Burkitt's lymphoma and nasopharyngeal carcinoma. Werne Henle, the son of a surgeon and grandson of the renowned anatomist Friedrich Gustav Jacob Henle of nephron loop fame, was born in Dortmund, Germany, in August 1910. He was educated at the Universities of Munich and Heidelberg, including graduation and internship in medicine. Classified as being 25% non-Aryan, Henle came to Philadelphia via a detour in Batista's Cuba. As an escapee from Nazi Germany, Henle was one of that cohort of refugee scientists, other professionals, and thinkers whom Jean Medawar and David Pyke refer to as "Hitler's Gift" to Britain and America.9 Werner married Gertrude, his sweetheart from their Heidelberg days, the day after she arrived in America in 1937. Together they were a formidable virological research duo at the Children's Hospital of Philadelphia until just a few months before Werner's death in July 1987.10 The Henles had shown that even an inactivated virus can call forth interference against both related and completely unrelated viruses when injected into embryonated eggs.7, 11, 12 Their work on the factors that govern viral interference culminated in a key review of its day,13 although unfortunately none of the hypotheses contained therein turned out to be true. Parenthetically, one should note that in the post-war United States both hens' eggs and research funding were relatively plentiful but were not so in Britain. But more on that later. In Japan in the 1950s, virologists Yasu-ichi Nagano and Yasuhiko Kojima of the Institute for Infectious Disease (now the Institute of Medical Science) at the University of Tokyo came tantalizingly close to identifying the facteur inhibiteur, as they called it, that rapidly inhibited viral replication only a few hours after rabbit skin is inoculated with an ultraviolet radiation-inactivated vaccinia virus and is challenged with the live version.14 After a series of thoughtfully conducted experiments, Nagano and Kojima could conclude, in French, that this inhibitory factor was distinct from both viral particles and immune serum,15, 16 but yet its identity remained elusive until the breakthrough by Isaacs and Lindenmann in the laboratories of the Medical Research Council's National Institute for Medical Research in Mill Hill, in the suburbs of London. Some authors17 have drawn parallels between the discovery of interferon and the discovery of the structure of DNA, which was published a few years earlier.18 Both advances were made in the 1950s in a Britain that was still recovering from the exhaustion of the Second World War, in fairly well-supported Medical Research Council laboratories, by duets made up of a seasoned "older man" from the host laboratory and a younger trainee from abroad. Both Alick Isaacs and Francis Crick were all of 36 years old, while Jean Lindenmann, a post-doctoral fellow from Switzerland, was 33, and James Watson from the United States was only 24 years old. However, whereas Watson and Crick were jointly awarded a Nobel Prize 9 years later for a discovery that received instantaneous recognition and approval, no such honor fell to Lindenmann and Isaacs. The Nobel Committee would had to have woken up to the importance of interferon far sooner to make the award, because Alick Isaacs died tragically just 10 years later in University College Hospital, London, where and when, by chance, the current author was a medical student. Unlike Watson or Crick, Alick Isaacs already enjoyed high prestige as a biomedical scientist when, in the summer of 1956, he and Jean Lindenmann embarked on the climactic experiments that would soon solve the riddle of viral interference that Isaacs had been pursuing for several years. Isaacs was born and academically bred in Glasgow, Scotland, where, despite having garnered many student prizes in clinical medicine, he was drawn to the laboratory and to microbiology. His fascination with virology was consummated during the 2 years that he spent at the Walter and Eliza Hall Institute of Medical Research in Melbourne, Australia, studying with Frank MacFarlane Burnet (1899-1985), who in 1960 shared the Nobel Prize in Medicine or Physiology with Peter Brian Medawar (1915-1987) for the discovery of acquired immunological tolerance. Medawar himself became Director of the Institute at Mill Hill a few years later (1962-1971). It was Burnet, in fact, in collaboration with W.I.B. Beveridge, who in the 1940s devised the enduring technique for cultivating viruses on the chorioallantoic membrane of embryonated chicken eggs. Lindenmann, who just recently has celebrated his 80th birthday, arrived at Mill Hill in July 1956 on a fellowship from the Swiss Academy of Medical Sciences to learn virology under C.H. Andrewes (later Sir Christopher). Isaacs, who had been in Mill Hill for a few years as well, also headed the World Influenza Centre that had been established there by the World Health Organization in 1947 as an international clearinghouse and reference center for research on the epidemiology of influenza globally. For Lindenmann, it soon became obvious that his task under Andrewes, to grow polio virus in primary rabbit kidney cells, was technically doomed, although it took several months to abandon that hopeless quest. Luckily, his future was soon to change. Lindenmann was introduced to Isaacs one teatime in early August 1956, and as soon as they both realized that they had a mutual interest in viral interference, a collaboration began.19 Lindenmann's as yet unpublished results showing that inactivated influenza readily induces interference in embryonated eggs even if the virus is attached to chick red cells20 were quickly confirmed21 using red cell ghosts and fragments of chorioallantoic membrane, as suggested by Isaacs. Isaacs reasoned that by substituting ghosts for intact red cells it might be possible to visualize by electron microscopy the viral component (likely nucleic acid) that evokes interference after entering the embryonic tissue.22 This was a miserable failure, but the use of chorioallantoic membrane fragments instead of whole eggs, an innovation that allowed ready separation of the susceptible tissue from anything that could be added to it,23 was the key to success. One suspects that the idea to use membrane fragments may have been driven as much by a lingering frugal mentality conditioned by years of wartime austerity in Britain as by science. Food rationing in Britain, which had been imposed for 14 years since January 1940 and included among its many deprivations an allowance of only 1 fresh egg per week for each citizen since June 1941, was finally ended in July 1954, just 2 years before the fateful experiments began. Luckily for their momentous teatime chat, it is noteworthy that tea rationing was removed 4 years earlier, in 1952. Irrespective of the justification for using membranes, each egg provided 7 or 8 different experimental points, and sometimes many more. In their experiments, it turned out that when membrane fragments were incubated at 37°C with a 56°C heat-inactivated Melbourne strain of influenza A, interfering activity appeared in the membranes after a lag of several hours and later was secreted into the medium.1 The surprising observation was that medium that was depleted of virus or interfering potential by incubation with membranes had as much, and not less, interference activity as the fresh virus-rich medium. To the prepared minds of Isaacs and Lindenmann, primed subconsciously perhaps by a speculation that Andrewes had made 14 years earlier when discussing viral interference in tissue culture, namely that an inhibitory substance might be generated,24 came the inescapable realization that they had actually found that inhibitory substance. The prevailing wisdom had been either that the internalized virus somehow blocks receptors, or uses up some essential foodstuff in the cell, or produces an accumulation of abortive viral particles that gum up the cell's replication machinery.2 Lindenmann referred to the interfering activity as "interferon," more as convenient laboratory shorthand than as a result of a deliberate taxonomic exercise.22 But the term stuck and later became accepted.25 It only remained to differentiate interferon from the virus itself or its components and to describe some of its properties, which they did in short order.2, 26 These later reports included demonstrations that like inactivated influenza27 interferon injected intradermally into rabbit skin together with vaccinia virus interferes locally with the development of the vaccinial lesion, and that interferon is probably proteinaceous since it is inactivated by trypsin.26 That these three trailblazing manuscripts1, 2, 26 were all submitted for publication within a year of that auspicious teatime tête-à-tête is testimony to the excitement, industry, and fervor of the authors. The reaction to the discovery of interferon was not universally enthusiastic at first. Between 1957 and 1965 only 107 papers were published with "interferon" in their titles.22 In some scientific quarters skepticism was caustic, as evidenced by such derogatory alternative terms for interferon as "imaginon"28 and "misinterpreton."29 The ups and downs of interferon research, its pharmaceutical development, clinical trials, prescribing, and marketing are legendary and have been the focus of many articles30-35 and monographs.36-38 Suffice it to say, interferon has weathered these storms and found a role in the treatment of viral infections, inflammatory and fibrotic disorders, and many malignancies.39 The discovery of the hepatitis B virus40 and the recognition that it frequently causes not only acute but also chronic contagion41 with resultant end-organ injury, cancer,42 and death, provided an ideal scenario for testing the therapeutic efficacy of our quintessential natural antiviral agent; namely, interferon. A bolster to this therapeutic rationale came from the observation by many,43-45 but not all,46 investigators that the circulatory interferon response that may be seen during other clinical viral infections47 is lacking in patients with viral hepatitis. Neither short-term48 nor long-term49 steroid treatment had been good for chronic hepatitis B, whereas results of pilot studies of leukocyte interferon given to 4 patients at Stanford University in California,50 1 patient in Leuven, and 2 chimpanzees in Antwerp, Belgium,51 demonstrated the efficacy of interferon in reducing viral replication, temporarily at least. Chimpanzees suffer from hepatitis B too.52 Although it was not the main focus of the Californian study, perhaps the most important point was seen in the utility of making measurements, in clinical trials, of markers of hepatitis B viral replication such as circulating viral DNA, DNA polymerase, e antigen, and even core antigen in the circulation and, in the Belgian study, in liver tissue. Naturally and appropriately these promising reports were met with skepticism concerning the purity of interferon derived from virus-infected human leukocytes, the mechanism of action, and the durability of the response.53 The use of adenosine arabinoside or its phosphorylated analog as antiviral agents to circumvent the problem of shortage of interferon was short-lived by reason of toxicity, inconvenience, and lack of efficacy.54 And although the preparation and production of interferon from human leukocytes, lymphoblastoid cells including Namalwa cells, fibroblasts, and other cell lines was progressively improved, purified, and upscaled,55-59 as so personably and engagingly retold by Kari Cantell,37 it was not for 10 years until the advent of recombinant techniques, which started with the successful cloning of a human interferon gene,60 that reliable clinical studies could be performed.61, 62 Nowadays, just as interferon therapy for hepatitis B has come of age, it is being challenged weekly, it seems, by an expanding array of new nucleoside and nucleotide analogs.63 The story of interferon therapy for the contagion of hepatitis C is at the same time both more adventurous and more successful than for hepatitis B. Long before the discovery of the hepatitis C virus was published in 198964 and screening to detect it was devised,65 the existence of another viral cause of acute and chronic hepatitis was known, and the clinical and epidemiological attributes of so-called non-A, non-B hepatitis were reasonably well defined.66, 67 To the prepared mind of Dr. Jay Hoofnagle at the National Institutes of Health in Bethesda, Maryland, this chronic viral liver disease called out for interferon therapy too, even though its viral cause had not yet been determined. Finding pharmaceutical companies to support the venture was not easy. But, drawing from a stable of chronic non-A, non-B hepatitis patients who were young, otherwise healthy, and had consistently high aminotransferase elevations, Hoofnagle and colleagues selected 10 for treatment in their landmark study.68 They gave decremental doses of recombinant human alpha interferon 2b over intervals for up to 12 months. In 8 of the 10 patients, elevated serum aminotransferase levels decreased rapidly during therapy and eventually fell to the normal or near normal range. This contrasted with the delayed response and enzyme flare that were seen in patients with hepatitis B who are treated with interferon.62 In two of the patients in whom interferon therapy was stopped after 4 months, there was a prompt relapse of enzyme elevation followed by an equally prompt return to normality when treatment was recommenced. Other groups soon followed suit and obtained similar results,69-71 and before long, a series of large multicenter randomized trials was conducted in many patients around the world, which established the role of interferon and ribavirin as the treatment of choice for chronic hepatitis C, as reviewed recently.72 The importance of the original pilot observation and results of subsequent registration trials was not lost on the lay press, for on Friday, November 30, 1989, the New York Times announced with enthusiasm, "Drug can control hepatitis C virus, researchers find." The tabloid National Enquirer had published an interview with Dr. Hoofnagle some 2 years earlier (Fig. 2), which, although accurate, raised some eyebrows among the higher echelons of the National Institutes of Health. The successful results of the pilot study of interferon treatment of hepatitis C received acclaim in the popular press. Dr. Hoofnagle's memorable interview was published in the National Enquirer. Subsequent testing of stored sera from the patients in the original pilot study confirmed that all had suffered from chronic hepatitis C infection.73 A 10-year follow-up study showed that the 5 patients who had achieved a 6-month sustained response after therapy remained hepatitis C RNA negative and had normal or near normal liver histology, whereas the other patients had symptoms of chronic hepatitis and 2 had shown severe disease progression.74 Many refinements to therapy and new approaches are on the horizon for patients with chronic hepatitis C.75 New agents are being tested, such as synthetic bacterial-like CpG-rich oligodeoxynucleotides that bind to the TLR9 receptor on dendritic cells to stimulate the immune system to mount a Th1 immunomodulatory antiviral response,76 which is reminiscent in a way of the use of interferon inducers almost 30 years ago in chronic hepatitis B–infected chimpanzees.77 Alick Isaacs, who would have been 84 years old now had he survived, would surely feel vindicated for his efforts if he could see the enormous impact of the discovery of imaginon/misinterpreton that he shared with Jean Lindenmann almost 50 years ago. Isaacs was elected a Fellow of the Royal Society a year before he died, in recognition of the discovery of interferon and his many other scientific contributions. The prepared mind of Jean Lindenmann has often been appreciated, not only for his share in the discovery of interferon and for his other scientific achievements, but for his gift as a commentator on the history and philosophy of science, and for his modesty and unassuming nature,78 and deservedly so. Most of all, the author is delighted that Jean Lindemann personally verified this brief account of the discovery of interferon. The author also thanks Dr. Samuel Baron for his reminiscences of working with Alick Isaacs and his time spent in Room 215 at the MRC National Institute for Medical Research. The author appreciates that Dr. Jay Hoofnagle related to him the history of the early days of interferon therapy for viral hepatitis. The author acknowledges Margie Myers for skillful manuscript preparation.