John Enders, PhD
(February 10, 1897 - September 8, 1985)
Born in the United States
Years of Discovery: 1949-1958
Father of Modern Vaccine Methods - And the Polio & Measles Vaccines

Virologist is virulent to viruses! John Enders was a virologist who, along with key researchers Thomas Weller and Frederick Robbins, pioneered techniques for studying viruses in the laboratory that revolutionized virology, leading directly to the development of vaccines against many serious diseases. At the late age of 50, he started a laboratory at Children's Hospital in Boston, after World War II. There, his team was the first to produce enough poliovirus in test tubes to be studied effectively. This work led to the vaccines developed by Jonas Salk and Albert Sabin that eradicated polio from the U.S. Although Salk and Sabin were the much more famous scientists, it was Enders' team who was awarded the Nobel Prize for Medicine or Physiology in 1954 for its work on culturing the poliovirus. After this, Enders made his greatest accomplishment when he led another team that created the measles vaccine, a vaccine that has saved well over 100 million lives.

Enders never rushed through life. He was a flight instructor in World War I when biplanes were made of canvas and had two wings. He graduated from Yale, and then tried his hand at business. Unhappy, he quit and returned to school at Harvard to get a graduate degree in English. Still passionless, he decided to get a doctorate in philology, the study of language in literature. But it wasn't fulfilling either. He wrote a friend, "I mouth the strange syllables of ten forgotten languages, letting my spirits fall, my youth pass. If this mood lasts, I shall by Heaven, throw it all to the four winds and go forth into the world like Faust, even if I have to bear his penalty." 

His life changed when he accompanied a friend to a lab. There, he was fascinated by the experiments and met a professor, Hans Zinsser, who would become his mentor. Enders described Zinsser as, "A man of superlative energy. Literature, politics, history, and science-all he discussed with spontaneity and without self-consciousness. Everything was illuminated by an apt allusion drawn from the most diverse sources, or by a witty tale. Voltaire seemed just around the corner, and Laurence Sterne upon the stair. . . . Under such influences, the laboratory became much more than a place just to work and teach; it became a way of life."

At the age of thirty, Enders entered the doctoral program in bacteriology at Harvard. After getting his doctorate he still didn't rise quickly rise in the academic world. Instead, he worked in Zinsser's lab until Zinsser died. Soon thereafter World War II altered academic research and then Enders' wife died. Picking up the pieces in 1945, he was offered a chance to run his own lab at Boston's Children's Hospital.

There, Enders and his colleagues sparked a revolution in the field of virology with the development of their tissue culture techniques. Viruses had been grown in tissue cultures prior to Enders, but not consistently  and with difficulty. Most researchers used live animals as the medium in which to grow viruses and, as a result, very little progress was made in virology.

Enders' team devised methods that allowed virologists to grow a limitless supply of many viruses in numerous tissues. They also demonstrated the value of looking for cell damage due to the invasion of the viruses. They built a toolbox of techniques to handle viruses in test tubes, drastically shortening the time required for experiments, and allowing for the testing of virtually endless ideas. Soon, their methods were used in laboratories all over the world, leading to numerous major discoveries.

When Enders began his work only 13 human disease-causing viruses had been isolated. By 1961, 58 more human viral pathogens had been cultured, plus another 300 viruses that infect animals. Finally, Enders' team demonstrated all the techniques necessary for each step in the creation of viral vaccines. Only 3 vaccines were available to children when Enders began his work. Today there are 18 recommended childhood vaccines and 4 adult vaccines.

• Vaccinations are one of the most important medical treatments ever devised. Better than a cure like penicillin, there is no suffering – 23 diseases are simply prevented.

Enders' biggest discovery was the development of the measles vaccine. His team spent the 1950s developing it, then handed it off to Maurice Hilleman and Merck Pharmaceuticals to finish. Measles is caused by a virus and is one of the most contagious diseases known. The virus normally grows in the cells lining the back of the throat and those lining the lungs. The first sign of infection is a high fever lasting one to seven days. During this initial stage the patient may develop multiple symptoms, including a runny nose, cough, red and watery eyes, and small white spots inside the cheeks. A rash develops after several days, typically beginning on the face and upper neck, and then spreading to the hands and feet. Poorly nourished children are at an increased risk of contracting a severe case of measles, especially those who have a vitamin A deficiency or whose immune system is compromised. Childhood deaths are usually caused by the complications associated with measles. The most serious complications include blindness, an infection of the brain and spinal cord (encephalitis), severe diarrhea, ear infections and severe respiratory infections, such as pneumonia, which is the most common cause of death associated with measles. Due to widespread use of the measles vaccine, U.S. cases now range from the 10s to the low 1,000s. Vaccination has been the key.

And the measles vaccine is used worldwide. An estimated 85% of the world's population now get a measles vaccine in childhood.  As a result, there has been a significant reduction in estimated global measles deaths. In 2000 it was estimated that  6,000,000 people a year died from measles, mostly children.  Now in 2021 that number is down to 120,000, all due to the measles vaccine.  

Enders is also remembered for his personal charm and keen interest in all knowledge. He maintained close professional relationships with his associates, and mentored and furthered the careers of many fellow scientists. His spirit of personal and scientific collaboration fostered the important discoveries that saved millions of lives and improved the health of billions of people, especially children, across the world.

Written by science writer, Elizabeth Downs

Lives Saved: Over 131,000,000

Read Ender's Feature Chapter in the Book

Book Excerpt
Fame or Lack Thereof
Working on a measles vaccine, they had great difficulty.  Month after month these attempts proved unsuccessful, so time after time Enders and his team left the lab without making progress. Enders had the habit of taking a taxi home from the lab and got to know many of the drivers. One evening, on the trip home, the taxi driver asked Enders, "How is the progress of your research?" Enders told him it wasn't progressing well. The driver encouraged him to keep at it and, "Perhaps you will discover something wonderful, like Dr. Salk."(Note that it was Enders and his team that made the discovery that made possible Dr. Salk's polio vaccine). 


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When Enders began his work, more than 6 million people died from measles each year. In 2019, the worldwide number was under 500.

Both polio and measles are considered to be eradicable, meaning that they can be completely eliminated from the world. This requires humans being the only host for the disease. If that happens, no one will ever get sick or die again and after a while no vaccine will even be necessary. 

Today polio is only endemic in two countries - Pakistan, and Afghanistan

Quotes By John Enders
"This antipodal revolution of my studies has been of large value in helping me to obtain that Pisgah sight of things and people that perhaps is the ultimate aim of my apparently inconsistent, faltering and obscure action." -On his changing his PhD to science, referencing Pisgah, the mountain which Moses ascended to see the Promised Land

"In these experiments the tissue culture method was employed with uncertain results. But the conviction was gained that it represented a basic tool for the study of viruses of which the possible applications were almost unlimited."
-Enders discussing his research

"From time to time we had considered the mounting evidence...in favor of the possibility that these agents (of polio) might not be strict neurotropes.... Such ideas were in our minds when the decision was taken to use a mixture of human embryonic skin and muscle tissue in suspended cell cultures in the hope that the virus of varicella might multiply in the cells of its natural host.... while close at hand in the storage cabinet was the Lansing strain of poliomyelitis virus. Thereupon it suddenly occurred to us that everything had been prepared almost without conscious effort on our part for a new attempt to cultivate the agent in extraneural tissue."
-Nobel Prize Speech

"At this point it seems appropriate to remark that the discovery of the antibiotics has, as in so many other areas, worked a revolution in the field of tissue culture. Through the use of these substances it is now not only possible to apply tissue cultures to the routine isolation of viruses from materials heavily contaminated with microorganisms, but it has become feasible to use them under conditions and in numbers which in the past would have been quite unthinkable. Here then we have another example of how one discovery leads to many others often of quite a different nature."
-Nobel Prize, discussing the importance of antibiotics in their own research

"From these observations we concluded that, as with other viral agents, the virulence of poliomyelitis virus is not a fixed attribute but on the contrary may readily be altered under appropriate conditions."
-Nobel Prize Speech, explaining in understated words that a vaccine was possible by weakening the polio virus until it could be used as a vaccine!

Quotes About John Enders
"The superficial Enders guise was that of a round-shouldered, overburdened, sometimes meek, pedantic scientist - a caricature he deliberately fostered and exploited effectively as a shield against the unwelcome intrusion of assignments to distracting committees or administrative chores. In fact, he was a strong, competitive, thoroughly contemporary, artful academician who conserved his energies for those challenges he judged worthy."
-Samuel Katz, who worked with Enders during the research to develop a measles vaccine and knew him well

"The achievement of Enders, Weller, and Robbins was the starting point not only of modern polio-virology, but it launched the revolution rightly called molecular virology."
-Hans J. Eggers, MD

"He favored doing small-scale experiments and reviewing the results of each as they came out to define another simple question and plan the next experiment to answer it."
-David Tyrrell, virologist

"The door was always open to visitors from throughout the world. Few scientists left such a visit unaccompanied by carefully packaged boxes containing samples of virus, cells, sera, reagents, or other ingredients, to ensure the ready progress of their own experiments back at home. The data in laboratory notebooks were shared with visitors who sought specific information on experiments recently completed or still underway.... His philosophy was that the more people working on a problem, the sooner a solution would be found."
-Samuel Katz

"Dr. Enders pitched a very long forward pass, and I happened to be in the right place to receive it."
-Jonas Salk, on Ender's team's laying the foundation for the polio vaccine

Enders was "firmly convinced that the size or magnificence of laboratory surroundings and equipment had little relationship to productivity or success - if anything an inverse relationship."
-Samuel Katz

"His relations with the staff were such that they were able, and indeed encouraged, to produce suggestions for the next experiment or a new idea to solve a problem. This would be discussed from all angles and quite often the chief would put forward a different idea, but in such a way that the trainee was glad to take it up rather than feeling resentfully that he had been forced to abandon his own idea and accept that of ‘the Boss.'"
-David Tyrrell, virologist

Vaccines are Ubiquitous & Omnipresent Throughout Our Lives
It is bizarre that vaccines are controversial, when virtually everyone has had vaccines.
The average person born today may get 70+ vaccines in their lifetime. 
There are 31 childhood vaccines (counting booster shots). Over 90% of people have received some of these vaccines.
Did you know that all beef, poultry, and pork you eat comes from vaccinated animals?
And that all horses, dogs, cats, and zoo animals are vaccinated?
Who wants to live in a world where you can’t pet your friends’ dog because it may not have been vaccinated, and may have rabies?

Anecdotes
Enders' Teaching Style
After receiving his doctorate in bacteriology from Harvard, Enders became an instructor and laboratory scientist at Harvard. As a teacher, he was noted for his personal magnetism in small-group and one-on-one settings, including in the lab, but seemed less suited to giving lectures. "Delivery was another matter," said a later colleague, Samuel Katz. "After adjusting the microphone around his neck and checking that it was live, he would extract a large handkerchief from his vest pocket and blow his nose with significant amplification by the sound system. We were never certain whether that was a nervous habit or meant deliberately to arouse the students' attention or laughter, but it usually did both. He extracted a large gold pocket watch from another compartment of his vest and placed it on the lectern to assure punctuality in his presentation. The lecture was articulate, but dry, and rarely overwhelmed the audience. His own disciples sat squirming in the back row seats hoping the students would perceive the gems they were receiving. After the lecture, we joined him for the short walk back to the laboratory in the old Carnegie Building of Children's Hospital, assuring him of how fine his presentation had been, even though both he and we suspected that few of the students appreciated what had been offered."

Formative Years
A frequent visitor to Enders' family home when he was a child wore a white suit. It was Samuel Clemens, better known as Mark Twain.

He matriculated to Yale University, but interrupted his education to enlist in the Naval Reserve to serve in World War I. There he learned to pilot the fragile, rickety bi-planes of the time. Air flight was only fifteen years old, and these daring pilots sat in cold, open cockpits. Enders was not sent to combat, but became a flight instructor in Pensacola, Florida. If he had any notions of being a daredevil himself, his experiences flying completely cured him. He decided he had used up all his luck in the air, and forever after was extremely reluctant to fly.

Enders lived with medical students in Mrs. Patch's comfortable boarding house. There he met an animated Australian, Hugh Ward, whose daily description of his doctoral work in microbiology piqued Enders' interest. "We soon became friends," Enders wrote, "and thus I fell into the habit of going to the laboratory with him in the evening and watching him work. I became increasingly fascinated by the subject-which manifestly gave him so much pleasure and about which he talked with such enthusiasm."

The early 1940s greatly upset Enders' idyllic lab life. In September of 1940 Hans Zinsser died, not only costing Enders his cherished mentor, but also burdening him with the lab's bureaucratic administrative duties, which were definitely not his forte. Then, World War II began, resulting in most of the young doctoral candidates leaving for military service, thus ending many lines of research. Tragedy struck again in 1943, when Enders' wife, Sarah, suddenly died from acute myocarditis, an inflammation of the heart.

In the Lab
After the war, Enders was asked to establish a research laboratory on infectious diseases at Boston Children's Hospital, which was located just across the street from Harvard Medical School, and had various ties to Harvard. Today, that may sound like an odd place to do research, but in the 1940s it was ideal. Researchers were drawn to pediatrics, because children were both the battleground and the living laboratories of infectious microorganisms like polio and measles. Enders kept several novelty hats on a shelf in his office, some of which he had collected and others which had been given to him. When exciting experiments were to be examined, he would put one on.

Enders and his colleagues knew well they were dealing with a dangerous pathogen in polio, and had already developed safety procedures for handling viral specimens. Weller didn't even want his wife, Kay, to visit the lab. To be extra cautious, they put two monkeys they had autopsied at the animal building into heavy bags, lugged them back to the lab, and put them into the autoclave. Cooking the monkeys in it would kill any virus that remained alive so they could be disposed. The whole floor stank.

During this time, the Enders' team was writing papers and giving frequent presentations of its findings, so many researchers would visit. Jonas Salk was one. Katz says, "The door was always open to visitors from throughout the world. 

Death Bed
On a September evening at their water front home in Connecticut, in 1985, Enders was reading T.S. Eliot aloud to his wife, Carolyn. He finished and went to bed, then quietly died. He was eighty-eight. At his memorial service his friend, the Bishop F.C. Laurence, said, "John Enders never lost his sense of wonder - wonder at the great mystery that exists and surrounds all of God's creation. This awareness is what gave him his wide vision and open mindedness, his continued interest in all things new, his ability to listen, his humility in the presence of this great mystery, and his never-ending search for the truth." His widow said that John briefly revealed his heart when he told her, concerning how creation ran, "There must be a mind behind it all."

Enders' Laboratory Was Like a Family
The Enders' laboratory began as a group of diverse researchers, but grew, literally, into a family. Shortly after Frederick Robbins joined the lab he fell in love with a laboratory technician, Alice Northrop. Soon they married. Enders followed suit, marrying Carolyn Keane, another laboratory worker, in 1951. Robbins writes that Carolyn's, "cheerful disposition, facility in social intercourse, and great energy proved to be of enormous support." Fred Robbins thought so much of Enders that he suggested to Alice that they name their second daughter after him. She agreed, and the name Louise Enders Robbins was entered upon the birth certificate.
A well-regarded tradition in the laboratory was the family Christmas party. Enders donned a crimson smoking jacket and plied his guests with drinks. Katz recalls, "One delightful tradition involved the drawing of names from a hat, a few weeks before the holiday, for the giving of Christmas presents. More attention was focused on the requisite poem which accompanied the gift, and which was read aloud at the time of presentation, than on the gift itself. Some of the efforts were indeed remarkable, but none matched those which Enders himself composed, and it was a true joy to be the recipient of his poem and gift. The evening ended with the Chief at the piano and his guests gathered round him, his "family" singing Christmas Carols."

Alice Robbins (lab tech) - remembrances email from March 7, 2008
"Dr. Enders had unusually broad interests and enjoyed discussions in the lab at lunch about all sorts of things as well as work: politics, literature, the arts, even exciting sailing adventures. The laboratory space was small, only four rooms--two labs, Dr. Enders' office, and a glassware washing room. Because Dr. Enders did not wish to eat at the hospital, which Tom Weller always did, the rest of us (Jeanette Levens, Carol Keane, Fred and I) had lunch at the big table in the large lab adjacent to Dr. Enders' office. Jeanette, Carol, and I made the lunch, being careful not to have anything containing egg as Dr. Enders was allergic to eggs. "Does it have egg in it, Alice?" he would say in a worried voice. Those lunch times and the conversations were fascinating and quite the highlight of the day for me.

It was a close-knit group because when Dr. Enders set up the lab, he wanted to have people around him that he knew. Besides Jeanette, his long-time technician, and Tom and Fred, whom he had known since they were medical students, he hired Carolyn Keane (always known as Carol) to be his secretary and me to be Tom's technician. He had known both of us at the Bacteriology Department at the medical school. We all liked each other and admired Dr. Enders, whom we called the Boss. Dr. Enders liked to know about our activities but he didn't really like my going off skiing on weekends. He used to see me off with the comment (jokingly) "If you break something, just be sure to break your leg and not your arm."

Dr. Enders had a relaxed manner, and coming in in the morning later than the rest of us, after having been served tea at home by his old family retainer, Bridget, who padded around in tennis shoes and called him "the master," he would often say, "I've been thinking this morning, Fred," and then describe an idea for an experiment. With his imaginative mind, charm, inquisitiveness, and soft-spoken manner, Dr. Enders, in his old well-loved tweed jackets, created an atmosphere in the lab which made it a very special place, memories of which we treasure."

Colleague Samuel Katz in an email dated March 4, 2008
"Working with Enders explains his uniquely wonderful role as a mentor for us. He deliberately enrolled only 4 or 5 fellows at a time in order to have sufficient interactions with them. He was a renaissance scholar, interested in all knowledge, not just medical science. He sought research productivity for its benefits to all mankind, not for personal recognition. He was generous of his time, knowledge and friendship, but penurious of public funds (the only NIH grantee to return funds to them at the end of a fiscal year). He never held press conferences, but our research results were published in carefully worded manuscripts in journals with critical editorial review. We gave virus, cells, sera to any legitimate investigator who visited to observe our research, because his philosophy was that the more people working on a problem, the sooner a solution would be found. I could go on and on. Obviously he was my father-figure as well as my mentor!"

Personal Information
Early Life
John Enders was born on February 10, 1897, into a family of high achievers. His grandfather walked from town to town selling insurance, eventually becoming president of the Aetna Insurance Company. His father was president of Hartford National Bank. A frequent family visitor during Enders' childhood wore a spotless white suit and was better known by his pseudonym - Mark Twain. In school, Enders had trouble with math and physics, and later said that during those years he "preferred in the main certain of the so-called humanities - Latin, French, German, and English literature, although biological subjects always proved highly attractive."

He matriculated at Yale University, but interrupted his education to enlist in the Naval Reserve to serve in World War I. There he learned to pilot the fragile, rickety bi-planes of the time. Enders was not sent to combat, but became a flight instructor in Pensacola, Florida. If he had any notions of being a daredevil himself, his flying experiences completely cured him. He decided he had used up all his luck in the air, and forever after was extremely reluctant to fly.

Education: A Late Start in Science

After graduating from Yale, Enders set out to uphold the family tradition of business. He brokered real estate for a while, but had trouble seeing the value of convincing someone to buy something they already wanted. After four uninspired years in business, Enders quit and went back to school.

Choosing Harvard, he received a masters degree in English, then decided to pursue a doctorate in philology, the study of language used in literature. He couldn't get excited about it either, writing a friend, "I mouth the strange syllables of ten forgotten languages, letting my spirits fall, my youth pass. If this mood lasts, I shall by Heaven, throw it all to the four winds and go forth into the world like Faust, even if I have to bear his penalty." While lost in purpose, Enders' life was far from miserable. He lived with some medical students in Mrs. Patch's comfortable boarding house. There he met an animated Australian, Hugh Ward, whose daily description of his doctoral work in microbiology piqued Enders' interest. "We soon became friends," Enders wrote, "and thus I fell into the habit of going to the laboratory with him in the evening and watching him work. I became increasingly fascinated by the subject-which manifestly gave him so much pleasure and about which he talked with such enthusiasm." Ward also introduced him to Hans Zinsser, a charismatic professor.

Together, Ward and Zinsser transformed Enders' life, convincing him that his intellect was best suited to science. So, in 1927, at the age of 30, considered late even in his day, Enders made a 180 degree turn in his studies and entered the doctoral program in bacteriology at Harvard. It was rare to earn a doctorate under Zinsser, a demanding taskmaster, but Enders earned his in 1930, with a thesis on anaphylactic shock caused by carbohydrates extracted from tubercular bacteria.

Family
In 1927, Enders married Sarah Frances Bennett. They had a son, John Ostrom Enders II, and daughter, Sarah. Tragically, his wife died in 1943 of acute myocarditis, an inflammation of the heart. In 1951 Enders married Carolyn Keane and gained a stepson, William Edmund Keane. They resided in Boston, Massachusetts and kept a summer home on Long Island sound.

Career Path
After earning his doctorate in 1930, Enders became an instructor and worked in the Harvard laboratories under Hans Zinsser. Enders showed little of the academic ambition typical of PhD's in their thirties. It was five years before he became an assistant professor, and another seven years before he became an associate professor. It took him thirteen years before he employed a personal technician. Research, as opposed to advancing his career, was his main interest.

When, in 1937, Harvard's experimental kittens came down with feline distemper, Enders and a young epidemiologist, William Hammon, began a careful study of the disease. They found it was caused by a virus, and worked on developing a vaccine. This and other studies turned Enders' interest toward viruses, where he made his first major breakthrough - "the development of serologic techniques for the detection of antibodies to the mumps virus."

After World War II, Enders was asked to establish a research laboratory on infectious diseases at Boston Children's Hospital, which was located just across the street from Harvard Medical School, and had various ties to Harvard. In 1940, Enders had worked with Thomas Weller, a young medical student, on an experiment that had revolutionary implications. Using a roller tube apparatus they had kept cowpox virus (vaccinia) growing for nine weeks. Previous viral life span in tissue culture had usually been measured in hours or days, not weeks. Now that Weller was back from military duty, Enders brought him on to be the assistant director and to take care of administrative duties.

Enders and Weller were allocated four rooms on the second floor of the Carnegie Building, a three-story building next to Harvard's coal-fired power plant. Two rooms were turned into labs, one room was the glass washroom, and the final room was Enders' office. Animals were kept a block away in another building. Enders and Weller furnished the lab with the standard equipment, such as an autoclave to sterilize equipment and chemical hoods, along with some more novel items. Embryonated hens' eggs had recently been introduced as media in which to grow viruses, so they purchased an egg incubator and visualizing lights to shine into the eggs. Early in 1948, they added Frederick Robbins, Weller's college roommate to the team.

The aim of the lab, which many thought futile, was to study numerous viruses in tissue cultures. Enders had already worked with viruses such as herpes simplex, influenza, measles, and mumps, and wanted the new lab to expand such research. There were numerous experiments going on at any one time.

John Enders' Life: A Timeline
1896 - Born in West Hartford, Connecticut
1917 - World War I Naval Reserves flight instructor in Pensacola, Florida
1920 - Yale Graduate, B.A.
1921-1925 - Real Estate Broker
1925-1927 - Harvard MA, English
1927 - 1930 - Harvard PhD, Bacteriology
1930-1940 - Laboratory assistant in Hans Zinsser's lab, Harvard
1937 - With William Hammon, investigated distemper and worked on distemper vaccine
1939 - Developed serologic techniques for the detection of antibodies to the mumps virus
1940 - Revolutionary experiment that allowed viruses to grow in cell cultures for nine weeks
1943 - Wife Sarah died
1947 - Began laboratory at Boston's Children's Hospital with Thomas Weller
1948 - Frederick Robbins joined lab
1949 - Published revolutionary article on how to grow the poliovirus
1951 - Married Carolyn Keane
1952 - Jonas Salk developed the killed polio vaccine
1953 - Enders began research on measles
1954 - Enders, Weller and Robbins win the Nobel Prize for their work on polio
1956 - Became a full professor at Harvard
1957 - Albert Sabin developed the attenuated polio vaccine
1958 - Began testing a measles vaccine
1963 - Measles vaccine, improved by Maurice Hilleman at Merck, produced commercially
1985 - Died on September 8, at home, in Waterford, Connecticut, age 88

Key Insight
The key insight that revolutionized virology was learning how to grow viruses in test tubes for long lengths of time. Without being able to grow viruses, scientists can't study them, learn  if any medicine will attack them, and how to make vaccines to prevent them. 

Roller tube tissue cultures had been perfected by George Gey in 1933, but Enders had been one of the first to use them with viruses. Rather than growing animal tissue in flasks, researchers using roller tubes plant tissue cells in plasma clots on the sides of test tubes. Viruses can then be inoculated into this tissue. A nutritious fluid consisting of inorganic salts, serum from animals or humans, and an extract from embryonic tissue is supplied to keep the tissue alive. The capped test tubes are then kept almost horizontal and heated in an incubator to 37 degrees Centigrade (99°F.), which is the temperature of the human body. The test tube is slowly turned at a rate of eight to ten times an hour, exposing the tissue to both the liquid nutrient and air. The goal is to more accurately mimic conditions inside the human body, where tissue is exposed to an active environment of nutrients and waste product removal, rather than the static environment of a motionless test tube or flask. A big advantage of using the roller tube technique comes when viewing the cultures under a microscope. Tissue from flasks has to be chopped, and then prepared as slides for the microscope, while the outgrowth of cells into the plasma clot of a roller tube can be repeatedly examined in the living state, as the cells grow and the virus replicates.

After World War II, when Enders had his lab up and running, Thomas Weller used a technique that had some of the characteristics of a roller tube to keep a mumps virus alive for a long period of time. The team next kept poliovirus alive. The techniques the team built to keep viruses alive in test tubes completely changed virology, by allowing many viruses to be experimented upon endlessly.

The Science Behind the Discovery
Basic Science Primer: Problems in Virus Research

Viruses
Viruses are very different from bacteria, the other type of agent that causes most disease. They are so tiny that they were not even seen until the advent of the electron microscope in the 1930's, when Enders was beginning his research. Made up of genetic material -- either DNA or RNA strands -- and encapsulated in a protective protein shell, viruses thrive in bacteria, fungi, plant, and animal cells, co-opting the cells' genetic machinery to acquire energy to reproduce. It is arguable whether they are even life forms, but they definitely have a large impact in humans. When they parasitically infect our bodies, they cause many diseases, including the common cold, flu, hepatitis, herpes, HIV, SARS, Ebola, and covid just to name a few.  And unlike bacteria that may be cured with antibiotics, there are very few anti-viral drugs to attack these diseases once they take over our bodies.  So far, the only good way to attack them is to prevent them by vaccines.

The Challenge of Studying Viruses
The exceedingly small size of viruses (hundreds or thousands of viruses can exist inside a single human cell) makes them devilishly difficult to study. At the time Enders began to take an interest in them, most viruses could only be grown in vivo (Latin for "in the living"), which meant in living animals. Animal experiments are expensive, slow, and awkward - not to mention, they usually require sacrificing  the life of the animal when a scientist wants to observe the effect of a particular trial.

Scientists needed techniques for studying viruses in vitro (in test tubes), which presented several challenges. First, a given virus will only grow in certain cells from certain animals. Second, the cells must be healthy when infected with a virus, and a test tube is not a naturally healthy setting for a living cell. Third, the cells must remain healthy throughout the experiment, which means they must be provided with nutritious food. Fourth, the cells must remain uncontaminated but, in a test tube, bacteria, other viruses, and fungi can easily contaminate and kill cells. Finally, if a virus can be induced to multiply, it often kills the very cells into which it has been cultured, releasing viruses into the liquid remnants. The only way to maintain those viruses is to transfer them to a tissue culture of new cells - called a subculture. All of this has to be repeated over and over again. In the 1940s before Ender's team developed their techniques, growing viruses in test tubes in sufficient quantity to experiment with was a major barrier to virology research.

Enders' Research On Polio
Polio was the most feared disease in America from the 1920s through the 1950s. Polio came like clockwork when the weather warmed, and left with the first chill. Usually, the epidemic was mild and most of the victims, largely children, recovered without permanent damage. Every few years, however, it struck with particular viciousness. In 1916, New York City tallied 9,300 cases, with 2,200 children dying. Half the survivors were paralyzed. In this setting, an accurate idea of how polio reproduced in human tissue was crucial to advancing knowledge about it, so a vaccine or cure could be produced. Karl Landsteiner published the basic facts about polio in 1909. It is a virus, and monkeys were the experimental animals of choice, since it also infects them. There was - and still is - no cure.

Growth of Poliovirus in a Flask
Enders ran a collaborative laboratory. Samuel Katz, who worked with him on the measles vaccine, said, "Every day Enders took time to make the rounds of the laboratory benches to talk with each fellow and ask, ‘What's new?' That two-word question was a wonderfully effective stimulus to enhance laboratory productivity because a fresh answer to the inquiry earned extra personal time with the Chief to discuss one's observations."

With such a free flow of ideas, early in 1948 Thomas Weller set about trying to isolate the varicella virus, the cause of chicken pox and shingles, using embryonated hens' eggs. He failed. As a next experiment he decided to use the varicella virus. Enders had worked a lot with the mumps virus, so suggested that he try it next instead. Weller's idea was to build upon the experiment he had performed with Enders when he had been a student back in 1940. The experiment allowed them to keep cowpox virus alive for nine weeks using a roller tube apparatus in a flask, which was revolutionary. Next he set about attempting to grow viruses in a flask by modifying the Maitland technique, whereby tissue is placed in a flask along with a nutrient solution, then inoculated with a virus. He inoculated chicken eggs' amniotic membranes (the fluid sac that surrounds and protects the embryo) with the mumps virus, and placed it in a flask with a nutrient solution of balanced salt solution and ox serum ultrafiltrate. After a short time, the fluid containing the virus was drawn off and put into another flask with fresh tissue - making a subculture. Weller's idea was to modify this technique by leaving the tissue in the flask and replacing the nutrient fluid every 4 days. This allowed the tissue to remain alive for a longer period of time, giving the virus more time to grow in each cell. The experiment worked. The tissue did not quickly die, and the mumps virus continued to multiply and grow. Enders suggested they do the same experiment using an influenza virus. They isolated it as well, and found they could keep tissue growing for 30 days or longer.

Next, Weller prepared to use this technique to try to isolate the chicken pox virus, as he had originally intended. Since the varicella virus was only known to grow in human cells, he prepared cultures of skin and muscle tissue from aborted human embryonic tissue, minced them, and placed them into flasks. He inoculated four of them with cultures of throat washings from a child who had chicken pox. Another four flasks were not inoculated - they would act as controls to compare the results of the first four. He had enough skin and muscle culture left for four more flasks.

The lab had cultures of the Lansing strain of the polio virus and Weller put it in the four remaining flasks. He recalls, "It was almost as an afterthought: I was focusing on growing the varicella virus, not the poliovirus." He was disappointed - there was no evidence that the chicken pox virus was growing in the flasks. However, he took fluids of the cultures he had inoculated with poliovirus, and injected them into the brains of five mice. Within a week, one mouse became paralyzed and died. As the days passed, 3 more mice died.

Never before had there been evidence that poliovirus could be grown in non-nerve cells. In their Nobel lecture, the Enders team said, "...Thereupon it suddenly occurred to us that everything had been prepared almost without conscious effort on our part for a new attempt to cultivate the agent in extraneural tissue."
Further experiments showed they could grow the other two known types of polio, the Brunhilde and Leon strains, in the same manner. Enders and his colleagues demonstrated that the virus would grow in human embryonic tissues from the intestine, liver, kidney, adrenal, brain, heart, spleen, and lung. The Flexner polio paradigm, that polio could only be grown in nerve cells, was completely overturned.

Cytopathic Effect and Other Ways of Monitoring Viruses
Enders' previous work with the mumps virus had shown that fluids infected with the virus visibly affected red blood cells, which could be observed under a microscope. This provided an important lab test that could be used to tell if a fluid was really infected by the mumps virus. If he and his colleagues could find a similar test for the polio virus, showing that it infected and harmed tissue cells, it would be much easier than autopsying monkeys after inoculating them.

One day, peering into the microscope, Enders saw a poliovirus-infected cell explode. "Cytopathogenicity," he exclaimed, inventing a word. The word, truncated to cytopathic, later became common in virology. He and his team could observe the effect of the poliovirus on the cultured tissue cells 16 to 32 days after being inoculated.

Next, they found a chemical marker for the growth of the virus in infected tissue by adding phenol red, a dye. The poliovirus affected the cells' metabolism so that infected cells produced less acid than those not infected. In uninfected control cells, the dye changed color to yellow as the cells acidified. In infected cells, it remained red. Thus they had a ready test for the presence of polio in human tissues. The researchers also discovered a positive correlation between the acidity of the cultures inoculated with poliovirus and the amount of time since inoculation. Such precision allowed them to measure the infectivity of the virus as well as determine the minimum dose needed to infect various types of tissue.

The team eventually shortened their ability to recognize the virus to eight days and developed a test tube assay to determine the types of poliovirus and their respective antibodies in human or animal blood serum. These techniques would all become common testing techniques in virology.

The Roller Tube Apparatus
Roller tube tissue cultures had been perfected by George Gey in 1933 and Enders was one of the first to use them with viruses. Rather than growing animal tissue in flasks, researchers using roller tubes planted tissue on the sides of test tubes. The test tube is slowly rotated to mimic conditions inside the human body, where tissue is exposed to an active environment of nutrients and waste product removal, rather than the static environment of a motionless test tube or flask.

A big advantage of using the roller tube technique comes when viewing the cultures under a microscope. Tissue from flasks has to be prepared as slides for the microscope, whereas the outgrowth of cells in a roller tube can be repeatedly examined in the living state as the cells grow and the virus replicates.

A handy carpenter made the Enders researchers a roller tube apparatus with a round wooden holder of about 100 test tubes. When they used the roller tubes, they found that the virus grew more rapidly and to higher titres (concentrations) than in the suspended cultures in the flasks. This cut the time necessary to observe the growing virus to four days.

Identifying Poliovirus Infection in Animals and Humans Using Antibiotics in Culture - Leaving Monkeys at Play
Until the Enders laboratory found their new technique, scientists who wanted to isolate and type polio virus cultures could only do so by injecting the virus directly into the brains of living monkeys, letting the virus infect the brain tissue, killing the monkeys, and testing samples for virus. In the end, more than 100,000 monkeys were sacrificed in the development of an as-yet only dreamed-of polio vaccine. Enders and his team found a way to spare future monkeys by successfully isolating poliovirus from the monkeys' feces.

Frederick Robbins performed much of this research, adding penicillin and streptomycin - two newly discovered antibiotics - to the feces samples to kill off bacteria. Tissue cultures, inoculated with the purified samples, quickly showed the telltale signs of polio infection. They found that they could often isolate and type virus from feces within 48 hours in humans as well. Experiments demonstrated that many children carry viruses similar to polio, often with no symptoms. Within two years, the team had isolated and typed 13 strains of poliovirus. They also discovered the first of a whole group of viruses that became known as ECHO viruses (enteric cytopathic human orphan viruses), which are cousins to polio.

Weakening the Poliovirus to Make a Vaccine - Attenuation as the Holy Grail
A vaccine is a preventative medicine that jumpstarts the immune system into producing antibodies to attack a specific disease, usually before the immunized person contracts the disease itself. A virus that is devastating to humans can be passed serially through various non-human tissues and end up vastly weakened - known in virology as attenuated - yet retain enough of its original protein coat signature that the antibodies produced by the human immune system to fight the attenuated virus in the vaccine will also fight the original virus. A virus has to be grown over and over again in different media until it mutates into the desired weaker form.This "playing with evolution," a method developed by Louis Pasteur in the 1870's from his work with chicken cholera, anthrax, and rabies, is time-consuming and arduous.

In the Enders laboratory, Thomas Weller worked on propagating and attenuating the poliovirus. He grew the Lansing virus through 23 passages (a passage occurring whenever he changed the tissues in which the virus was growing, aka subcultures) over 331 days. He grew the Brunhilde strain -- the dominant cause of polio in humans -- through 15 passages over a period of 267 days. Periodically, the researchers tested it. They found that the virulence (or strength of the virus) had declined after three passages. As the number of passages increased, the virus' virulence decreased well over 100,000-fold. The team's conclusion was spelled out in their Nobel Prize lecture: "From these observations we concluded that, as with other viral agents, the virulence of poliomyelitis virus is not a fixed attribute but on the contrary may readily be altered under appropriate conditions." In less understated words - a vaccine was possible!

Enders' Team Published Their Findings in 1949 and Left the Polio Vaccine to Jonas Salk and Albert Sabin to Create
Interestingly, the Enders lab did not try to develop the polio vaccine itself, even though they had the knowledge and could have easily secured the funding. "Enders, in his thoughtful way," Robbins said, "felt that this was not the kind of work our laboratory was best suited for." So the team stepped back and passed the task to Jonas Salk and Albert Sabin, who already had big labs dedicated to vaccine development. Both had pretty much stalled before the breakthroughs in tissue culture made by Enders' team. Suddenly, with a way to obtain an endless supply of viruses, they made quick progress. Salk himself said, "Dr. Enders pitched a very long forward pass, and I happened to be in the right place to receive it."

In 1952, Salk developed a vaccine that contained chemically killed virus from all three types of polio. In what was then the largest public medical experiment in history, 200,000 children were injected with the Salk vaccine, and the vaccine proved almost 80 percent effective at preventing infection with polio. It came at a propitious time for, in 1952, polio again flared up. The Salk vaccine was licensed for use in 1955, and within the next decade, immunization made polio epidemics a thing of the past in most of the developed world.

In 1957, Albert Sabin developed a vaccine of weakened, or attenuated, virus, which he insisted was safer and more effective than the Salk killed vaccine, and which could be taken orally in delicious sugar cubes. Because it was oral, Sabin's vaccine had the additional advantage of causing the body to produce antibodies in the intestine as well as in the bloodstream. Therefore, unlike the killed vaccine, it prevented the intestines from serving as reservoirs for poliovirus. Nor did it require the booster shots that Salk's vaccine required. It was Sabin's live vaccine that became the standard vaccine used in the United States in 1961. Endorsed by the World Health Organization, it eventually was used throughout most of the world.

Measles Virus and Vaccine
Measles usually strikes in waves of two to four years and is astonishingly communicable-even the slightest contact will pass it on. It remains incurable to this day, although secondary infections can be treated with antibiotics. Measles was a common childhood disease in America into the 1960s, with almost every child becoming sick from it. Although not a big killer in America, thanks to our country's good nutrition and medical care, worldwide it was devastating. The World Health Organization estimates that when Enders began his work, 106 million people got measles each year, and more than 6 million - mostly children - died.

Enders formed a new team after his success with polio and worked through most of the 1950's on a measles vaccine. In 1960 he handed the vaccine off to Maurice Hilleman at the pharmaceutical company, Merck. There the vaccine was perfected. It came into widespread use in 1963 and, now being used all over the world, the number of measles deaths today is down to less than 150,000 a year.

Enders' Research - Measles
Pediatrician Louis Z. Cooper described it at that time: "Before a measles vaccine was available, more than nine out of ten children caught measles, half of them before they were five. Measles starts with a high fever, a runny nose, a cough, and a sore throat. After five days, a blotchy red rash develops. Usually the disease runs its course in another week, but sometimes there are complications. One measles patient in every six develops pneumonia or a serious ear infection. One in every thousand gets measles encephalitis, an inflammation of the brain that can cause paralysis, mental retardation, and even death. Far from being a harmless childhood disease, measles kills more children than any other acute infectious illness."

Measles had been a particularly difficult virus to work with because there was no good animal host on which to experiment. When Enders began researching measles in 1953, Asian monkeys were the only nonhuman species that had been found to be susceptible to the virus, and they sometimes became infected and sometimes did not. Without a reliable test animal, and the tissue culture technique still being relatively new, little progress had been made with the measles virus.

Enders sent Tom Peebles, a pediatric resident, out to draw blood and collect throat swabs from kids in Boston who had measles. Using roller tube techniques, they began trying to isolate and grow virus from their samples. They continued to grow the viruses in serial passages through these media. From four to ten days after they were inoculated, the tissues began to show the abnormal, cytopathic changes that indicated a virus had taken over the cells. The cells would develop multiple nuclei and become enlarged, then die. Over time, nearly all of the cells followed this path to destruction. They next combined human sera from twelve people who had had measles to the tissues to see if antibodies in their sera would inhibit the cytopathic process. It did. This meant they were dealing with the measles virus and that the human immune system reacted successfully to the virus. As early as 1954 the tissue culture techniques were producing great results.

Next, Enders and his colleagues inoculated monkeys with the virus they had grown after 1, 2, and 23 serial tissue passages. Just as previous researchers had found, the results varied - some monkeys showed symptoms and others did not. To try to solve this riddle, they examined their next set of experimental monkeys before running any trials on them, and found that 22 of 24 monkeys from three different laboratories already had measles antibodies in their blood. They didn't know how the laboratory monkeys had become contaminated with measles antibodies, but that did explain the variable results. It also allowed monkeys to be used as reliable test animals - once the contaminated ones were weeded out.

Growing Measles Virus in Chick Cells
The Enders lab tried to grow the measles virus in tissues other than humans, such as pig, cattle or chick cells. In order to make a vaccine it was important to find a non-primate tissue in which to grow the virus. Otherwise, there was a high risk of contamination by other agents that would be pathogenic to humans. For several years they were completely stymied. In the team's third year of working with measles, they observed an interesting phenomenon. After passaging the Edmonston strain 23 times through human kidney cells, then 14 times through human amnion cells, they noticed that some cells did not undergo the typical cytopathic change. Instead of enlarging and blowing up when infected, some cells became star- or spindle-shaped. As the researchers continued to passage the virus, these cells came to be the predominant manifestation of the virus.
The virus had obviously changed. Might it now grow in a chick cell? After passaging the virus 23 times through human kidney cells and then 28 times through human amnion cells, the researchers cultured it in chick embryo amnion cells, chosen in the hope they would be analogous to human amnion cells. Success! They could continue to passage the virus many more times through those cells and obtain the same cytopathic reaction as they had seen in human amnion cells. They next tried growing measles in other chick embryonic tissues, and that worked too. At long last, they had found a growth medium that might yield a viable vaccine.

Attenuation of Measles Virus
There are newer methods today, but up until this century, there was no definite recipe for growing a virus in foreign tissue or for developing a vaccine. Virologist Joel Warren noted, with a literary allusion John Enders would have relished, that it is not unlike the recipe found in Macbeth: "Round about the cauldron go, in the poison'd entrail throw...cool it with a baboon's blood, then the charm is firm and good."

One technique used in making a vaccine is to help evolution along by helping a virus multiply in various tissues until it weakens - called attenuation. In order to attenuate the measles virus, Enders and his team needed to put it through enough passages to weaken it so it would no longer infect humans, but still be strong enough to produce the viral antigens needed to create an immune reaction. The lab developed two potential recipes. The best was from the Edmonston strain of measles virus. It was produced after an additional 6 passages through chick embryos and 14 passages in chick cell cultures. Tests on monkeys showed that the attenuated virus differed from the infectious virus in two necessary ways. The weakened virus could multiply and cause infection in chick-cell cultures, but neither multiplied nor caused symptoms in monkeys. Most important, monkeys produced measles antibodies 15 to 23 days after inoculation, which indicated that their immune systems would fight off the wild measles virus.

Measles Vaccine Development
Enders' team now had to establish that their attenuated virus would work as a vaccine in humans, so medical doctors began tests on small groups of children. Between December of 1958 and March of 1960, they ran six sets of tests on children. The vaccine gave the children a mild infection but nothing worse, and did not make them contagious. More importantly, it gave children immunity from full-fledged measles. They published their results in eight papers in the New England Journal of Medicine in July, 1960. At a conference in New York, the eminent virologist and bacteriologist Joseph Smadel stood up and said "John, you've done it again."

The one negative effect of the vaccine was that 30 percent of the children in the trials came down with a fever as a result of the shots. Joseph Stokes, a pediatrician at the Children's Hospital in Philadelphia, developed the practical solution of giving shots of human gamma globulin, containing measles antibodies in one arm, and the measles vaccine in the other, but this was not going to work on a world-wide scale. So, as he had done with polio, Enders made his attenuated strain available to others to refine his work.

Here the vaccine developer Maurice Hilleman stepped in, and applied the resources of the pharmaceutical company Merck, Sharp, and Dohme (now the giant corporation, Merck). Hilleman discovered that Enders' strain had become contaminated with a chicken leukemia virus, and he found a breeder in California who had developed a line of chickens that were immune to the virus. Illustrating once again the importance of the serial passage technique, Merck's team passed Ender's measles virus 24 times through primary human kidney cells and 28 times through human amniotic cells, as Enders had done, but then upped it to 12 times through embryonated hens' eggs, and 57 times through chick embryo cells. The end result of Hilleman's labor, after passing 35 quality control tests, was a commercial vaccine licensed for use in 1963.

Enders later wrote that his work on the measles vaccine was the most personally satisfying and socially significant. Over the following decades, the measles vaccine spread all over the world. By 2000, more than 80 percent of the world's children were receiving it. By 2006, the number of measles cases had fallen from 106 million in 1963, to 20 million, and the death rate from over 6 million to 345,000.

Key Contributors to Polio Research
John Enders
Thomas Weller
Frederick Robbins

Key Contributors to the Measles Vaccine
John Enders
Maurice Hilleman
Samuel Katz
Thomas Peebles
Anna Mitus
Kevin McCarthy
Ann Holloway
Milan Milovanovic

Enders' Awards
1954 - Nobel Prize for Medicine: John Enders, Thomas Wells and Frederick Robbins, "for discovery of the ability of poliomyelitis to grow in cultures of various types of tissues."
1954 Lasker Award
1959 Time Man of the Year
1963 Presidential Medal of Freedom

Links
John F. Enders and the Measles Virus Vaccine - a Reminisensce, by Samuel Katz (Chapter 1)
https://www.google.com/books/edition/Measles/wZ41S3CKVPYC?hl=en&gbpv=1&dq=milan+milovanovic+measles&pg=PA5&printsec=frontcover 
Biographical Memoir by Thomas Weller and Frederick Robbins
https://www.nap.edu/read/6061/chapter/5
World Health Organization Measles Fact Sheet
http://www.who.int/mediacentre/factsheets/fs286/en/
John Ender's Wikipedia Page
https://en.wikipedia.org/wiki/John_Franklin_Enders 

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