Personal Reflections on Twentieth Century Vaccinology

SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH

PERSONAL REFLECTIONS ON TWENTIETH CENTURY VACCINOLOGY

Maurice R Hilleman

Merck Institute for Vaccinology, West Point, PA, USA

Abstract. The science of vaccinology was created in the late 18th and 19th centuries by “giants” of the time including Jenner, Pasteur, Koch, von Behring, Ehrlich and Lister. Relatively little technologic advance was made in the period leading to World War II except for yellow fever and influenza vaccines. Support for war efforts fueled developments which led to the modern era of vaccines of 1950 onwards. The author’s career in vaccinology, which began in 1944, was recognized in the 2002 Prince Mahidol Award given in Bangkok, Thailand. This review presents the substance of the Award lecture delivered on January 29, 2003.

INTRODUCTION rived from my birth and rearing on a farm in the state of Montana on the harsh Western Frontier Among the many pursuits of mankind, none where working with plants, animals, soil, mechan- can be more rewarding than that of preventing ics, and electricity provided a crucible for learn- diseases by procedures which derive from the ing, and a practical introduction to the workings practice of vaccinology. Vaccines, together with of the biological and physical sciences. Having sanitation and nutrition, have served as principal majored in science in high school, I went on to tools employed in Public Health to increase the receive a baccalaureate degree in microbiology health and lifespan of human beings. Public and chemistry from Montana State University in Health was a dedication of Prince Mahidol of 1941 and a doctorate from the University of Chi- Thailand whose memory is celebrated each year cago in 1944 in Medical Microbiology with spe- with the Prince Mahidol Awards in Medicine and cialization in virology. Knowledge of viruses was Public Health. We are reminded in this that clini- mostly a black box of disparate and anecdotal data cal medicine treats the individual, but public in the early 1940s, without even a textbook. My health treats the community to the benefit of all professor Francis Gordon and I did, however, pio- who reside therein. neer the first formal course in virology in the Present endeavors in the fields of USA, including both lecture and laboratory com- vaccinology, and the pursuits in basic science ponents. which make them possible, are enabled by the rich heritage of discoveries made by the great pio- INTRODUCTION TO INDUSTRY neers of the 18th and 19th century Europe, particu- larly by Jenner, Pasteur, Koch, von Behring, Having exhausted what university tenure had Ehrlich, and Lister, who laid down many of the to offer, I joined the Squibb Pharmaceutical Labo- basic principles and technologies we still follow ratories in New Jersey in 1944 to gain practical today. It has been the responsibility of 20th cen- and managerial experiences. I became the Head tury vaccine research both to serve that period of the Virus Laboratories, directing and conduct- and to provide a legacy for the 21st century. ing research, development, manufacturing, and quality control. Among our many labors, we pioneered and produced a Japanese B encephalitis EARLY BACKGROUND vaccine to protect military personnel in the Pa- My own interests in science and biology de- cific Theater of World War II, and we collabo- rated with Wendell Stanley in commercial devel- Correspondence: Maurice R Hilleman, Marck Institute opment of the Sharples centrifuge-purified influ- for Vaccinology, West Poit, PA 19118, USA. enza vaccine for military and civilian use.

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BASIC RESEARCH AT WALTER REED respiratory epithelium, which showed transmis- sible cytopathic change on inoculation of throat By 1948, I was convinced that my further samples from patients at Leonard Wood. This career should be focused on basic research but was the discovery of the adenoviruses, with which with need to take promising possibilities to prac- we could soon write a new chapter on epidemic tical realities. I joined the Walter Reed Army In- respiratory diseases. In the contemporary time stitute of Research in Washington, DC, in 1948 period, Dr Robert Huebner at the National Insti- as chief of respiratory diseases research, with tutes of Health found similar agents in cell cul- authority to explore them all but with special re- tures of tonsils and adenoids of children who were sponsibility to create a knowledge base for influ- latently infected but without evident disease. Viral enza viruses, and to avert the next pandemic of adaptation to monkey kidney cells allowed us to the disease. My first investigations were aimed develop a killed adenovirus vaccine that we at the study of antigenic heterogeneity of the vi- showed to be near 100% effective in preventing rus and to explain in full why the 1947-48 influ- adenovirus disease in two epidemics in the mili- enza epidemic was so severe and why the vac- tary. A killed vaccine for civilian use was licensed cine failed to protect. In comparing the antigenic in 1958. composition of previous and current virus isolates, I discovered progressive antigenic change, with BACK TO RESEARCH IN INDUSTRY time, which is now called antigenic drift, and is caused by point mutations needed by the virus to In 1957, after 10 years at Walter Reed, which evade the continuing evolvement of complemen- included a Sabbatical in 1951, as Visiting Inves- tary herd immunity. The 1947-48 epidemic was tigator at the Rockefeller Institute, I accepted an caused by virus of very marked antigenic drift. invitation to join the Merck Research Labs in West In 1957, when the World Health Organiza- Point, PA, to head a new Department of Virus and tion and our military surveillance systems failed Cell Biology to be formed. This new laboratory to detect, I noted a report in the press of a large was the brainchild of the famed Vannevar Bush, and severe outbreak of respiratory disease with who had become chairman of the company and high fever in Hong Kong. This had all the potents who believed that one day viruses would be of of an influenza pandemic and our findings in great scientific importance. strain analyses of specimens which we obtained A new pattern for industrial organization was from Japan allowed me to alert the national and evolved for the Department in which basic re- international communities to a pending pandemic search would serve as the foundation. I was given which would begin in the US with the opening of full responsibility, with authority and with ac- school in September. It did occur as predicted countability, to plan and direct all basic research and was named the Asian influenza pandemic of and development, clinical investigations, engi- 1957. With such early alert, we were able to en- neering scale-up, and government relations, tak- gage the manufacturers early enough to produce ing products, when developed, to manufacture and 40 million doses of vaccine by year’s end, avert- licensure with continued responsibility to main- ing many cases of influenza in the population. tain the products on the market. This centralized In 1951, I conducted a large study of influ- operation, while an overwhelming task, did pro- enza at Fort Leonard Wood MO. to collect throat vide a smoothly operating machine in which plan- samples from patients with the intent to determine ning and decisions were quick to evolve and whether there was antigenic change in the virus implement, and to pioneer new vaccines. Clini- on egg adaptation. Inadvertent shift in etiology cal working alliances were made with Dr Joseph in the overall epidemic left me with specimens Stokes in the Department of Pediatrics, and from a different disease that was of unknown eti- Children’s Hospital, at the University of Penn- ology and which gave an opportunity to explore. sylvania, and with Dr Victor Villarejos at the Loui- With availability of a trachea from a newly de- siana State Medical Research Unit in San Jose, ceased soldier, we prepared explant cultures of Costa Rica. Above all, the central focus through-

Vol 34 No. 2 June 2003 245 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH out was on basic research with progression to tar- tion, immunogenicity, protective efficacy, and geted developmental research on what appeared proof of safety studies in thousands of suscep- promising. tible child volunteers, with informed consent. Individual problems with measles vaccine MODERN ERA VACCINOLOGY involved elimination of indigenous chicken leu- kemia virus contamination, attenuation and The beginning of the modern era of achievement of further attenuation Ð first by co- vaccinology arguably may be fixed as 1950 with administration of immune globulin, and later by Enders’ breakthrough in tissue culture technol- further attenuation of virus itself. ogy for growing polioviruses. Our first project was to develop a purified and precisely standard- Mumps vaccine required finding a wild vi- ized killed polio vaccine which would correct the rus free of neurovirulence from which an appro- deficiencies of the Salk polio vaccine. This vac- priately attenuated vaccine virus could be devel- cine was licensed in 1960 under the name of oped. PURIVAX. Rubella vaccine relied on our finding of A major problem with poliovirus vaccine means to grow and attenuate the virus in duck production lays in the presence, in Macacus mon- cell culture, and in proving safety on contact of key kidney cell cultures, of more than 40 differ- vaccinated children with pregnant susceptible ent endogenous contaminating viruses. Seeking women. remedy, we grew our viruses in Cercopithecus Varicella vaccine proved difficult to attenu- monkey kidney cell cultures which proved to be ate, while retaining potency, and the OKA strain free of contaminating viruses. In using these cul- was substituted for our virus in the vaccine. tures, we discovered hitherto undetectable viruses Acceptable monovalent, bivalent, and triva- which grew and were detectable in Cercopithecus lent vaccines were developed and licensed be- cells. One such agent was the SV40, monkey tween 1963 and 1971. polyomavirus, which we also found to produce cancers, when inoculated into newborn hamsters. HEPATITIS B These findings, which provided means to remove contaminants, were important in making live po- The breakthrough discovery of hepatitis B lio vaccine possible, in simplifying killed virus surface antigen in the bloods of hepatitis poliovaccine production, in providing an early carriers by Blumberg opened the door to a vac- model both to study and understand virus-caused cine. We found vaccine development to be fea- cancer, and to elucidate the immunology of can- sible by applying a 4-step purification and inacti- cer. SV40 virus became a principal model for vation process of carrier plasma which involved studies of DNA virus-caused cancer, and a tool antigen purification and concentration by zonal for basic studies in molecular biology. centrifugal separation, followed by digestion with pepsin, urea dissociation and re-association, and PEDIATRIC LIVE VIRUS VACCINES treatment with formaldehyde. The pepsin, urea, and formaldehyde steps, individually, were able In 1957, it was perceived that live attenu- to destroy all detectable life forms and collectively ated vaccines might be developed to protect constituted a process to develop a vaccine which against measles, mumps, rubella and varicella in was safe for man. Following demonstration of children. These were only dreams at the time. safety and protective efficacy in chimpanzees, of Developing these vaccines faced common hurdles safety and potency for man, and determination which involved etiologic discovery, and propa- of a vaccine regimen, two controlled clinical stud- gation and attenuation to achieve acceptable clini- ies were carried out which showed essentially cal reactogenicity while retaining adequate po- 100% efficacy. This plasma-derived vaccine was tency. Most important, was the lack of animal the world’s first viral subunit vaccine and the first models and the need to conduct all basic attenua- licensed vaccine against any human cancer.

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The need for a more reliable source for hepa- to develop a vaccine against meningococcal dis- titis antigen led us, in 1975, to commission the ease to provide a solution to the drug resistance preparation of an expression vector to produce problem which had arisen against meningococ- surface antigen in yeast culture. The process was cal meningitis of military personnel. Polysaccha- optimized in our laboratories and a vaccine of ride antigens were extracted from meningococ- equal potency and safety was prepared when the cal types A, C, Y and WI35 organisms and used recombinant antigen was substituted for the to prepare vaccines which were safe and immu- plasma-derived antigen in the preparation. Clini- nogenic. They were licensed as single and biva- cal performance was the same and the vaccine lent preparations between 1974 and 1977. The was licensed in 1986. It represented the world’s quadrivalent vaccine was licensed in 1982. first recombinant-produced vaccine. Having entered the bacterial polysaccharide arena, we developed 14 and 23 valent pneumo- HEPATITIS A coccal vaccines which were licensed in 1977 and 1983, respectively. Purified polysaccharide vac- Hepatitis A vaccine was pioneered in our cine against Haemophilus influenzae was eagerly laboratories, with the first isolation of true hepa- pursued by a number of pharmaceutical compa- titis A virus in marmoset monkeys in 1973. Vi- nies following discovery that coupling of their rus that was purified from infected livers was used polysaccharides with proteins elicited T helper to develop tests to define the virus, the clinical as well as humoral antibody immune responses. picture, and the epidemiology of hepatitis A. Such Our product was licensed in 1989. purified virus was also used to create a prototype killed vaccine in 1978, which was highly effec- VIRAL INFLUENZA tive in preventing infection in vaccinated mar- mosets following challenge with live virus. Our Toxicity due to impurities in killed influenza breakthrough cultivation of the virus in cell cul- vaccine led us to develop a filter-purified and a ture in 1979 opened the door to a killed vaccine zonal gradient-purified vaccine in 1969 and 1970, for man, which copied the process of the proto- respectively. These refinements vastly reduced type marmoset liver vaccine. It was licensed in the reactogenicity of killed influenza vaccine. 1996. INTERFERON AND INTERFERON MAREKS NEURAL LYMPHOMATOSIS INDUCERS

Our research with SV40 virus had piqued One of our earliest programs of research at our interest to develop the world’s first licensed Merck, lays with purification and characteriza- anticancer vaccine. We accomplished this using tion of interferon following Isaac’s and Burmester’s live turkey herpes virus which was Lindenmann’s discovery of the substance in 1957. apathogenic in chickens but immunized against Chicken allantoic fluid interferon induced by neural lymphomatosis, called Marek’s disease, cultivation of influenza virus in embryonated when given to newly hatched chicks. A whole hens’ eggs, was purified 4,500-fold and had an infected cell Mareks vaccine was licensed in 1971 activity of 42 ten thousandths microgram of pro- and a purified dried virus vaccine was licensed in tein per unit of activity. The purified interferon 1975. The vaccine proved highly effective in pre- was shown to be a monomeric glycoprotein of venting tumor, death, and declining egg produc- 7,900 molecular weight (also multimeric). It was tion. It revolutionized the economics of the poul- the first of the cell cytokines to be discovered and try industry. characterized, and serves to control viral infection in nature. MENINGOCOCCI, PNEUMOCOCCI, Because of species-specificity of interferon, HAEMOPHILUS INFLUENZAE chicken interferon had no application to man. We We were asked in 1971 by the US military set about, therefore, to develop useable interferon

Vol 34 No. 2 June 2003 247 SOUTHEAST ASIAN J TROP MED PUBLIC HEALTH inducers for direct application in man by trying 20th century, though not well exploited, will be to find out how viruses accomplished induction. the heritage to the 21st century. These include the We found, in three different approaches, that sciences of vectors, adjuvants, recombinant ex- double-stranded RNA is foreign to cells and pro- pression, and the fledgling discoveries in genet- vides the danger signal for a cell to express anti- ics, genomics, proteomics and computation sci- viral interferon. Interferon induction is now rec- ence which will be importantly used. New live ognized as an early response effector substance attenuated vaccines, and subunit bacterial vac- elicited by the innate immune system in response cines will also be developed. to viral exposure. We found homopolymeric syn- Vaccinology is a field in which dreams may thetic poly I:C to induce interferon on injection be turned into realities. It is an activity which is into man. It is of particular interest as a pattern heavily overshadowed by uncertainties, but can recognition signal to the innate immune system. be conquered by persistent rational pursuits and by selective choices needed to surmount the hills BENEFITS FROM VACCINE and mountains in the quest. I would add finally APPLICATION that vaccine progress can best be made in close The accomplishments of the 20th century cooperation between government, academia, and vaccine research have proved to be of worldwide industry. He or she who wishes to engage in vac- significance. Its vaccines are now finding their cine research will be served by being well posi- way increasingly from the developed world into tioned into all three of these enterprises. routine use in the developing nations. Before vaccine, eight million persons worldwide died of REFERENCES measles per year. The annual mortality rate is now less than one million. Combined MMR, Hilleman MR. Six decades of vaccine development. where amply applied, has essentially eliminated Nature Med:Vaccine Suppl1998; 4: 507-14. clinical disease caused by these agents, and con- Hilleman MR. Personal historical chronicle of six de- genital rubella syndrome is now a rarity. Cost cades of basic and applied research in virology, savings from use of MMR in the US alone, in immunology, and vaccinology. Immunol Rev 1992, came to 5.1 billion dollars. Hepatitis B 1999; 170: 7-27. vaccine, now given routinely to newborn babies Hilleman MR. Vaccines in historic evolution and per- in more than 100 nations, has drastically reduced spective: a narrative of vaccine discoveries. Vac- the incidence of hepatitis B virus carriers with cine 2000; 18: 1436-47. major reduction in cirrhosis and cancer of the liver in countries where the vaccine has been exten- Hilleman MR. Overview of vaccinology in historic and future perspective. The whence and whither sively applied. of a dynamic science with complex dimensions. HERITAGE In: Ertl HJC, ed. DNA vaccines. Ch 1. New York: Kluwer Academic/Plenum Publishers Many of the technical breakthroughs in the 2003: 1-38.

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