The Flu in Retrospect: Etiology and Immunization

Influenza and Its Etiology

The etymology of disease names often reflects outdated concepts about the disease. Malaria was so named because it was supposedly caused by bad air. Influenza was thought to have an even higher origin––the influence of the stars, which was the cause of epidemics (3). Influenza is an acute, highly contagious, systemic disease producing headache, fever, chills, myalgia, and malaise, accompanied by respiratory symptoms, mainly cough and sore throat. In many cases, its onset is so abrupt that the patient can recall the exact minute of onset. In most cases, fever is present, ranging from 38–41 °C. The clinical presentations cover a wide spectrum, from mild, afebrile illness similar to the common cold, to illnesses with severe prostration yet few respiratory symptoms (4). Influenza is also notable for occurring in epidemic waves.

Influenza has posed an especially difficult problem in epidemiology and etiology. Its symptoms do not readily differentiate it from other infections, and the disease seems to vary from epidemic to epidemic. In addition, a number of secondary bacterial infections confuse the picture. Finally, for a long period of time, no suitable animal could be experimentally infected. The discovery in the 1930s that pigs suffer from a very similar disease, and that influenza can be transmitted to ferrets, opened the door to discovery of the viral cause of the infection. Earlier research concentrated on searching for a putative bacterial cause (5).

Pfeiffer’s Bacillus and Other Bacteria

The German physician and bacteriologist Richard Pfeiffer (1858–1945) was a student of Robert Koch, and later, became Koch’s assistant. Pfeiffer was one of the pioneers of typhoid vaccination and he described the phenomenon of bacteriolysis, further recognizing that the breakdown of some bacteria led to the release of heat-stable proteins (i.e., bacterial endotoxins) responsible for effecting diseases (6). (Indeed, the presence of endotoxins is the reason why perfectly sterile solutions may cause fever if injected intravenously.)

In 1892, Pfeiffer, then the scientific head of the Institute of Infectious Diseases in Berlin, isolated a new organism from secretions of influenza patients (5). Certain that this was the cause of influenza, he named it Bacillus (later Hemophilus) influenzae. H. influenzae is a very small (0.2–0.3 μm × 0.5–2 μm) Gram-negative bacillus which occurs singly, in pairs, or in short chains. It is non-motile and has no spores. It is difficult to grow in culture, and highly sensitive to light, heat, and drying (5).

With the discovery of what came to be called Pfeiffer’s bacillus, it seemed that the etiology of influenza had been established. During the disastrous flu pandemic of 1918 researchers around the world sought Pfeiffer’s bacillus in the secretions of patients, in hopes of preparing a protective antiserum or vaccine. The bacillus was found only in a minority of patients. At first, failure to find H. influenzae was attributed to technical difficulties, but eventually it became evident that this bacterium was not the cause of influenza.

In 1918, Peter Olitsky and Frederick Gates at Rockefeller Institute discovered, in the nasal secretions of influenza patients, a bacillus so tiny that it passed through a porcelain filter. The flu virus may actually have been present in that filtrate, but Olitsky and Gates believed the bacterium, which they named Dialister pneumosintes, to be the cause of influenza. It is a Gram-negative, anaerobic, non-motile, non-sporulent rod. It plays no role in influenza, but a search reveals that it has been isolated from subgingival plaque, and thus may be involved in periodontal disease (7). It has also been found in some human brain abscesses (8) and is suspected in other infections.

Besides H. influenzae and Dialister, other organisms––for example Streptococcus, Neisseria catarrhalis, and various types of Pneumococcus––have been named as causes of influenza, but all are now deemed to be secondary invaders (5). It was during the 1930s that the bacterial theories of influenza causation were laid to rest, and the fundamental experiments that established its viral etiology were carried out.

Discovering the Viral Culprit

During the great influenza epidemic of 1918, there appeared, particularly in eastern Iowa, a swine disease closely resembling human influenza. J. S. Koen, a veterinarian with the US Bureau of Animal Husbandry, first recognized it as a clinical entity and named it “hog flu” (9).

The Roaring Twenties were exceedingly quiet as regards progress on hog flu, but in 1931 Richard E. Shope at the Rockefeller Institute published the results of extensive studies of this disease. Working with secretions from sick Iowan hogs, he induced hog flu in healthy animals by intranasal administration of the material. The secretions had been passed through bacteria-retaining ceramic filters, proving that a virus must be the disease-causing agent. During three epizootic (i.e., epidemics occurring in animal populations) periods of hog flu, in which the disease was clinically the same but of varying severity and mortality, Shope found eight strains of the virus (9).

Early in 1933, a flu epidemic was spreading throughout London. William Smith, Christopher Andrewes, and P. P. Laidlaw, of the National Institute for Medical Research at Mill Hill, were convinced that the cause was viral and were working with sterile-filtered nasal washings from ill patients. Their key discovery was that human influenza could be transmitted to ferrets. Nasal washings from two recovered patients and from one person suffering from the common cold were non-infective. Serum from a recovered ferret neutralized the virus’s infectivity, indicating that antibodies were formed during the illness (10).

Smith’s group also investigated the relationship of their newly discovered virus to that of hog flu. They obtained samples of the latter from Shope in the US and found that it produced in ferrets a disease identical to that produced by the human virus (10).

It is noteworthy that both Shope and Smith found it necessary to determine whether Pfeiffer’s bacillus could act as an auxiliary cause of influenza. Shope found that H. influenzae (suis), isolated from pigs, did not by itself produce hog flu. He also provided these cultures to the British group, who found that the bacillus did not produce influenza in ferrets (9, 10).

Later in the decade, further fundamental discoveries were made. The great American virologist Thomas Francis, Jr. (Box 1) isolated influenza virus Type A in 1934 and a new strain, Type B, in 1940. Influenza virus was grown in tissue culture and on the chorioallantoic membrane of the developing chick embryo (until recently the only medium for production of influenza vaccine). The hypothesis that the influenza virus can mutate gained currency. Serum antibody tests and complement fixation tests were developed. In 1941, red-cell agglutination testing beame a useful tool (11). Then, in 1939, a medical school senior named Jonas Salk began working on influenza virus under Thomas Francis, and was introduced to the methodology of virus research.

Jonas Salk: From Bronx to Ann Arbor

Jonas Salk, a boy from the Bronx and the son of immigrant parents, entered New York University College of Medicine in 1934. He apparently knew even then that his calling lay in scientific research rather than medical practice. His brilliance was such that, after his first year, chemistry professor R. Keith Cannan suggested he take a one-year leave of absence, under a fellowship, to study biochemistry. He concentrated on learning protein chemistry and returned to medical school in 1936. In the senior year, each student was allowed two months for elective work. Salk chose to work under Thomas Francis, Jr., chairman of the bacteriology department (12).

As Francis told it later, he and Salk grew influenza virus at the medical school laboratory. Salk then carried the virus to the Rockefeller Institute, where George Lavin subjected it to UV radiation and further testing. In 1940, Salk, with Lavin and Francis as co-authors, published his first paper describing the effect of UV on the virus’s antigenic potency. An accidental discovery Salk made during this time but published later was that influenza virus could be concentrated, and thus partially purified, by adsorption on calcium phosphate at neutral pH (13).

Salk continued to worked with Francis, who secured for him a $100 per month grant from Rockefeller, until entering upon his internship in March 1940 at Mount Sinai Hospital in New York. Even though his main interest was basic research, his empathy for the suffering of people led him to become an outstanding intern and a fine clinician. Upon completing his internship, he sought a fellowship which would allow him to study human immunity to viral diseases. He was turned down at Rockefeller and other institutions; even the recommendation of Dr. Francis was unable to overcome the prejudices which were still considered respectable at that time (12).

Meanwhile, Francis had become head of the Department of Epidemiology at University of Michigan School of Public Health, working on both influenza and polio. He invited Salk to join him, offering grant support in the amount of $2,100 per annum from the National Research Council and the National Foundation for Infantile Paralysis. Even though he yearned to live in New York, Salk could not turn down this opportunity, arriving at Ann Arbor in April 1942.

Developing Influenza Vaccine for the Army

The armed forces were being built up at a rapid rate through enlistments and selective service. With large numbers of young men concentrated at military bases, on warships, and on active battle fronts, the commanders had to face the possibility of epidemics, especially influenza epidemics.

The 1918 pandemic had taken a severe toll on the American military. It was necessary to prevent such losses from recurring.

Before he came to Michigan, Francis had been appointed director of the Commission on Influenza of the US Army Epidemiological Board. When Salk joined him in Ann Arbor, he was assigned to work on influenza exclusively, under Francis’s tutelage. Later, in 1943, when Francis was given additional resposibilities by the Army, Salk became acting director of the Army’s Influenza Commission. The papers he authored or co-authored between 1942 and late 1947, when he left for Pittsburgh, show the scope of the research conducted at Ann Arbor. The topics include influenza epidemiology, laboratory diagnosis, methods for estimating virus concentration and antibody titer, stability of the red-cell agglutinating factor, vaccine standardization, clinical testing of vaccine, and adverse reactions to vaccines. Even during the 1950s at Pittsburgh, he published papers on influenza, including studies on the use of adjuvants in vaccines.

Just as an army sets up sentinel posts to prevent enemy infiltration, so did the Influenza Commission set up early detection posts at various military bases in the US. This enabled Salk’s group to detect an outbreak of type A virus at its onset in Michigan, before the disease was spotted elsewhere in the country (14). In spring 1943, Salk traveled to Fort Custer, MI, to investigate an outbreak of atypical pneumonia. He found that it was caused by the type A Weiss strain of influenza and included that strain in a vaccine which was to be field tested in winter of 1943–1944 (12).

The vaccine being developed was tested for efficacy on male residents of certain wards at Ypsilanti State Hospital, a mental institution located near Ann Arbor. Vaccines prepared from type A and type B viruses were tested separately. Both induced resistance to challenge with active virus. The vaccines were best if administered two weeks prior to exposure, and the effect was greatly reduced after 4.5 months (15, 16). Informed consent was not a consideration in that era; these men were certainly not able to provide or refuse it.

Scale-Up: Salk, Sharp & Dohme, and Webster

Once efficacy had been confirmed, the next step was to prepare a combined vaccine for large-scale field testing. To prepare the vaccine, virus was grown in embryonated hens’ eggs. The extra-embryonic fluid from each egg was removed by syringe and pooled. The virus was then concentrated and treated with formaldehyde 1:5000, which inactivated its infectivity while retaining its antigenic properties. After sterilization by filtration, a bacteriostatic preservative was added. The vaccine was filled into 50 mL multi-dose vials. For the clinical trial, an inactive control solution was prepared, composed of normal saline, formaldehyde, and preservative (17).

Once this procedure was worked out in Salk’s laboratory, it was necessary to prepare the production-scale quantities needed for administration to thousands of men. To obtain these supplies the Army contracted with Sharp & Dohme (S&D), a maker of pharmaceuticals and biologicals located in Glenolden, a suburb of Philadelphia. Later acquired by Merck, in the forties S&D was a major manufacturer of vaccines as well as blood plasma. Unsurprisingly, scale-up proved to be a problem. The labor-intensive process involved inoculating myriads of eggs individually and, after incubation, drawing off the allantoic fluid, all under aseptic conditions. It may have been S&D’s first experience with an egg-based process, as the antitoxins and toxoids of that time (e.g., diphtheria and tetanus) were made from horse serum. The people at S&D contacted Salk, who came east to work with them.

For two weeks in the summer of 1942, Salk remained at Glenolden, working mainly with Willard Webster (Box 2), a research assistant in S&D’s new Virus Research Department. What makes these two weeks notable is that we have first-person comments from both men about their collaboration. These materials were graciously made available to this author by Webster’s widow. Thus, in the 1995 newspaper interview cited above (1), Webster described Salk as follows: “He was a bundle of energy. He was most energetic, and most understanding and most helpful. Whenever he saw an area where he thought he could contribute, that was certainly the way he progressed.” More specific information is found in a memoir of his life written by Willard Webster (18):

Salk’s feeling toward Webster was expressed in a 1988 letter, after Connaught Laboratories dedicated the influenza building at their Swiftwater, PA facility in Webster’s honor. Salk, then attending an international AIDS conference, wrote to Webster from Stockholm:

“Dear Willard:

I have just learned of the well-deserved honor and recall vividly our work together in the summer of 1942 in the preparation of the first batch of influenza virus vaccine field tested that year. I shall always recall the pleasure of working with you and Betty Lee Hampil on what, in retrospect, was an historic event” (18).

Salk, with his intimate knowledge of his vaccine, and Webster, with his creativity in solving equipment and production problems, succeeded in scaling up the production process. Sharp & Dohme delivered the required supplies to the Army, and the clinical trial was enabled to proceed.

The First Influenza Vaccine

The Army’s controlled trial got under way in the fall of 1943, under the direction of Thomas Francis. The subjects were about 12,500 men in units of the Army Specialized Training Program at universities and at medical/dental schools in different areas of the US. The study was double-blind, with active and placebo alternated among the subjects. Concurrently, in mid-November an epidemic of type A influenza broke out in the Midwest, soon spreading to other sections of the country. This epidemic was mild, the illness generally lasting 3–4 days with a low incidence of complications. Among those inoculated the influenza attack rates were as follows:

Thus, the first influenza virus vaccine was proven to be effective (17) an accomplishment made before Salk’s––thirtieth birthday.

There can be no doubt that the experience Salk gained through his wartime work on influenza was key to his later triumph in the battle against polio. In addition, his influenza vaccine has been the template on which influenza vaccines have been built in the succeeding decades, and on which we base our hope for blunting the effects of the current H1N1 virus.