Salk’s Polio Vaccine Research Paper (1953)

Presented below are some of the highlights from an 18-page report by Jonas Salk that was provided as evidence for the successful vaccination against Polio. It is a rather long read so for editing purposes, information regarding the inactivation of “virus” by chemical means were edited out as well as some of the details on theoretical antibody responses. Feel free to read the full paper linked below for the entire picture.

Remember when reading this that no complete purification/isolation of any Polio “virus” has ever taken place to date yet they were attempting in 1953 to create a vaccine for a hypothetical boogeyman they had never directly seen nor characterized. The Salk vaccine was pulled off the market 8 years after it was introduced as it was considered too dangerous for causing Polio in tens of thousands, paralyzing hundreds and killing at least 10 and after being found to be contaminated with SV40, a monkey “virus” which was said to come from the monkey kidney tissue cultures used for the creation of the vaccine. This, in turn, led to the use of Sabin’s oral vaccine which was even more dangerous. The Polio vaccines were (and still are) causing the disease they were supposed to prevent:

STUDIES IN HUMAN SUBJECTS ON ACTIVE IMMUNIZATION AGAINST POLIOMYELITIS

HISTORICAL BACKGROUND

Immunologic Complexity of the Poliomyelitis Virus. Although the virus of poliomyelitis was first discovered by Landsteiner in 1909, it was not until the report of Burnet and Macnamara in 1931 that suspicion was aroused concerning the existence of more than one immunologic variety. Similar observations were described by Paul and Trask in 1933. Little attention was paid to these very important observations, however, until considerably later. In the meanwhile the first attempts at immunization of man were made by Brodie and Kolmer. Were it not for the fact that these attempts made in the early 1930’s were discontinued because the preparations employed were found unsafe for human use, failure might have resulted for other reasons, one of which was the lack of more precise elucidation of the immunologic nature of the virus; this has been accomplished only recently.

The surmise that more than one immunologic type of poliomyelitis virus exists was further supported by the studies of Lennette and Gordon in 1940 and of Kessel and his co-workers in 1946. Further confirmation of the results of these early investigations, and the firm establishment of the existence of at least two distinct types, was reported by Kessel and Pait in 1948 and by Bodian, Morgan, and Howe jn 1949. Soon thereafter both groups of investigators reported the existence of a third immunologic variety.

Because of the importance of the problem, a systematic program for immunologic classification of the poliomyelitis virus was organized under the Committee on Typing of the National Foundation for Infantile Paralysis. Through the cooperative effort of investigators at four universities, a study was undertaken to determine whether only three types exist, and to obtain preliminary information on the relative importance of the different immunologic varieties as a cause of the paralytic form of this disease. On the basis of the findings in the study of the first 100 strains examined, it appears that the development of a procedure for the control of poliomyelitis by immunologic means need be concerned only with the three varieties of the virus that have been found thus far. They are now referred to as type 1 for those that are related to the Brunhilde prototype; as type 2 for those that resemble the Lansing strain; and as type 3 for those that resemble the Leon strain.

Pathogenesis and Immunogenesis of Poliomyelitis.— A great many significant observations that bear on the subject of pathogenesis and immunogenesis have been made in the course of numerous epidemiological and laboratory investigations in recent years. Rather than recount these completely or chronologically, it would seem to be more pertinent to the present thesis to begin with one of the most significant of the recent discoveries, not only to indicate the way in which earlier views were changed but to point up the influence of this discovery on the course of subsequent research.

The discovery by Enders, Weller, and Robbins and of Evans and his associates that the virus of poliomyelitis could be propagated in cultures of non-neural
tissue not only made available new techniques for development of a source of virus for immunologic studies but influenced profoundly and brought evidence to bear on the question of whether the poliomyelitis virus is strictly neuronotropic. This discovery supported the hypothesis that the virus may multiply in non-neural tissue and only thereafter be disseminated from the portal of entry or site of multiplication to the central nervous system. This hypothesis suggests further that the paralytic sequel of infection might be avoided if an antibody barrier could be created in the blood stream to intercept the virus before invasion of the central nervous system. Perhaps the first decisive experimental demonstration that this might be possible was the observation by Morgan that antibody induced by subcutaneous vaccination of monkeys with live virus reduced the frequency of paralysis in animals subsequently inoculated intramuscularly with fully potent virus. Similar observations were made with a variety of strains in the course of the program on immunologic classification.

Another significant observation was the finding of virus in the blood stream prior to the onset of paralysis both in experimental animals and in man, which was reported independently by Bodian and Horstmann. This, too, added support to the hypothesis that the viruses of poliomyelitis are not strictly neuronotropic. Bodian promptly demonstrated that the virus could be intercepted before invasion of the central nervous system by passive administration of relatively low levels of antibody to cynomolgus monkeys and to chimpanzees that were then fed a virus that causes paralysis when given orally.

Further indication that viremia may represent a stage in the pathogenesis of central nervous system involvement in humans and that this may be prevented by the presence of a sufficient concentration of antibody in the serum was quickly forthcoming in the significant observation of Hammon and his associates. They showed that relatively small amounts of antibody reduced the incidence and severity of paralysis if passively administered to children prior to the exposure or early enough in the incubation period.

Epidemiological Aspects of Immunity in Poliomyelitis.—The enormous amount of epidemiologic data that has accumulated over the years has been interpreted by most investigators as indicating that paralysis is the accidental occurrence in the course of a systemic infection that is widely experienced and is usually accompanied by the development of immunity rather than by central nervous system damage. Such observations made in the field as well as in the laboratory are in keeping with the opinion long held that the reduced frequency of paralysis with advancing years is due, in all likelihood, to the acquisition of immunity following nonparalytic infection early in life. The recent observation by Sabin on the relationship of age of mice to the development of paralysis provides the basis for an attractive hypothesis to explain why crippling occurs less frequently and less severely in younger persons. Thus, it appears that, if immunity is not acquired in the early years when concomitant paralysis is less likely to occur, then the risk of paralysis some time in life is increased.

The reasonable immunologic explanation for the once mysterious epdemiological behavior of poliomyelitis is now well established. Only recently, however, has it been possible to begin a detailed and quantitative elucidation of the activities of the poliomyelitis virus under natural circumstances. Such systematic investigations can now be carried out through the application of Enders’ tissue culture technique not only for the isolation of virus but also for the quantitative estimation of antibody for all three immunologic types. It is now possible to determine with relative ease not only the immunologic identity of the infecting virus but the frequency of the occurrence of antibody for the three virus types in various population groups. These methods have facilitated the progress of the studies reported here by providing a simple technique for determining the effect of active immunization on the level of serum antibody.

APPROACHES TO ARTIFICIAL IMMUNIZATION

Among the earliest reports on immunization of monkeys and then of man were those of Brodie and of Kolmer6 in the early 1930’s. Their studies were conducted before precise knowledge regarding pathogenesis and immunologic complexity were available, and before there had accumulated the vast experience with the many strains that possess different pathogenic characteristics. Moreover, methods had not been developed for adequate purification of virus from suspensions of central nervous system tissue, nor were the dynamics of virus inactivation or modification well enough understood.

More than a decade later, Morgan clearly demonstrated that a formaldehyde-treated suspension of central nervous system tissue from monkeys containing a type 2 strain of poliomyelitis virus did, after a rather rigorous schedule of immunization, induce the formation of appreciable quantities of antibody. She was also able to induce a measurable degree of resistance to intracerebral challenge in monkeys vaccinated repeatedly with similarly prepared type 1 virus.

Other investigators in studies with mice and cotton rats confirmed the fact that preparations of type 2 virus that were noninfectious were still capable of inducing immunity in rodents. Thus, it would appear that materials devoid of demonstrable infectious activity for animals will immunize if given repeatedly in sufficient amount. This approach was extended by Howe, who was able to demonstrate antibody formation in chimpanzees. In parallel experiments in six human subjects, he observed the development of antibody that in most instances persisted over the observation period of six months; each child received 3.5 ml. of human gamma globulin followed by 3 ml. of vaccine.

Another approach that has been suggested for inducing immunity is that of the feeding of attenuated living virus. Koprowski and his associates have recently reported the results of studies in 20 human subjects fed a preparation of type 2 virus, which had been propagated in the central nervous system of rodents. In their studies, an immunologic response was induced and evidence found of virus multiplication in the gastrointestinal tract. The agent used for feeding was reported to be less pathogenic for the monkey than for rodents, but still caused paralysis when inoculated intracerebrally into the monkey.

Although the objective to be achieved, in terms of serum antibody level, is more clearly defined and more reasonable of attainment, the question that still remains
is how antibody formation may be induced in a practicable manner. The basic prerequisites are simple enough. These are first, a rich source of virus reasonably free of extraneous antigenic material and second, a method for destroying pathogenicity without completely destroying antigenic capacity. To these might be added a third, a means for enhancing antigenic activity if the richest source of virus available after treatment for destroying pathogenicity proves to be inadequate or borderline in its effectiveness. It is the purpose of this report to show how these objectives are being approached, and to present data on the degree of antibody response observed in man in the first experiments cautiously undertaken.

Virus of the three immunologic types used in these studies was derived from cultures of monkey testicular and monkey kidney tissue. The destruction of infectivity was accomplished by treatment with formaldehyde, and in some experiments emulsification with mineral oil employing the technique of Freund was used for enhancing antigenic activity. It now remains for studies in progress (1) to establish the limits of reproducibility of the conditions for preparing noninfectious vaccines and (2) to determine the degree of persistence in humans of antibody induced by such preparations. Ultimately it will be necessary to test the ability of virus-neutralizing substances in the circulating blood that have been induced by vaccination to protect against the paralytic consequences of contact with the different immunologic types of poliomyelitis virus under natural circumstances. This will be necessary because of the possibility that invasion of the central nervous system by certain strains that occur in nature at different times may not be via the blood stream but by direct extension along nerve pathways, even though the weight of evidence has shifted from the latter to the former as a result of observations in recent years.

It is evident from the foregoing that many observations made both in the laboratory and in the field have for a long time encouraged the expectation that immunization against poliomyelitis should be possible. The greater acceptability now of the idea that invasion of the central nervous system may be primarily via the blood stream rather than primarily along neural pathways has increased the likelihood of such a solution. Moreover, the knowledge now available has reduced to reasonable levels the preliminary requirements expected for the performance of an immunologie method.

MATERIAL AND METHODS

Source of Virus for Vaccine. —The vaccines employed in these studies were prepared from virus propagated in roller tube m cultures of two different tissues derived from monkeys. Monkey rather than human tissue was decided on for producing the virus for these experiments in human subjects because of the remote possibility that human tissue might occasionally be contaminated with such agents as the virus of infectious hepatitis that might conceivably propagate in cultures of human tissue. Even though the plan was to treat the culture fluids with formaldehyde to destroy the disease-producing property of the poliomyelitis virus, there could be no assurance that conditions for destruction of the latter, for which specific tests are available, would simultaneously destroy other agents of human disease for which no simple laboratory tests are available. Another consideration was the fact that methods had been worked out for producing the virus in reasonably high concentration employing relatively small amounts of monkey tissue.

Although testicular tissue was the first monkey tissue used for culturing the virus, it appeared desirable to investigate other monkey tissues for comparison. Enders and associates had employed such human sources as the foreskin, embryonic skin and muscle, the kidney, and the uterus. It seemed of interest, therefore, to explore cultures of monkey kidney and muscle, which are available in much larger supply than testicular tissue. The data in table 1 indicate the relative concentrations of virus derived from cultures of the kidney, testis, muscle, and liver of a single monkey. The advantage of the testicular cultures in yielding virus for long periods is offset by the greater abundance of virus in kidney cultures in the early days after virus inoculation. This example is presented to illustrate why the decision was made to use kidney rather than any of the other tissues in further studies on vaccines. The striking difference between cultures of kidney tissue and testicular tissue might well be due to the nature of the principal cell type, which is epithelial-like in kidney cultures and fibroblastic in testicular and muscle cultures.

In the search for a fluid medium free of protein and still satisfactory for maintaining the viability of the tissues and for supporting virus multiplication, it was soon found that the chemically defined mixture no. 199 described by Morgan, Morton, and Parker and first used for similar studies by Rhodes and his associates provided such a nutrient fluid. This mixture consists of a large number of amino acids, certain nucleic acid constituents, vitamins, and minerals, as well as other essential growth-promoting or growth-sustaining substances. Details of the use of this medium for cultivation of the three types of poliomyelitis virus in roller tube cultures will be elaborated on elsewhere. The roller cultures in which virus was propagated for the experiments to be described contained approximately 100 mg. of minced tissue with 4 ml. of nutrient fluid.

Selection of Strains.—For preparing the vaccines to be used in these investigations, strains were selected quite arbitrarily and principally on the basis of the regularity with which they could be propagated in relatively high titer in tissue culture. It is highly probable that substitutions will be made for certain of the strains now in use as further information is gathered in regard to the characteristics most desirable for inclusion in a vaccine.

The strains selected to represent types 1, 2, and 3, respectively, are known as the Mahoney, MEF-1, and Saukett strains. The Mahoney strain, which was isolate originally by Francis, was adapted to tissue culture from monkey central nervous system tissue. The MEF-1 strain, which was isolated by Schlessinger and Olitsky, was transferred to tissue culture from the spinal cord of paralyzed mice. The Saukett strain was isolated in this laboratory by direct inoculation of tissue culture with a fecal specimen from a patient paralyzed in 1950.

When inoculated into cynomolgus monkeys, these strains were found to have the following characteristics: The Mahoney strain is highly pathogenic by any route, whereas the MEF-1 and Saukett strains are essentially nonpathogenic except when given intracerebrally. When undiluted tissue culture fluids containing high concentrations of either of the latter two are injected by nonneural routes, paralysis will occur infrequently. Of the two, the Saukett strain produces the milder disease, even when given intracerebrally.”

Safety Tests.—The first problem that had to be resolved before human subjects could be inoculated was the question of safety. It has been the consensus that tests for safety should include the intracerebral inoculation of monkeys. In all of the material employed for human beings, the absence of infectivity for the cynomolgus monkey was established in prior tests by the intracerebral inoculation of 0.5 ml. of fluid in 6 to 10 animals. As an additional safeguard, the only preparations considered satisfactory for use were those that had been exposed to the action of formaldehyde for at least 24 to 48 hours after the test just described indicated that infectivity had been destroyed. The fact that amounts of virus that produce paralysis when given intracerebrally are not paralytogenic when given intramuscularly or intravenously suggests that the intracerebral safety test may provide a more than adequate margin in certifying a given preparation as safe for use in human subjects.”

“The mineral oil used in these experiments, known as “drakeol no. 6,” is manufactured under conditions that satisfy the requirements for medicinal oils as established by the National Formulary. The preparation of purified emulsifying agent was mixed as a 3% solution in the mineral oil and sterilized by Seitz filtration. This was then mixed with equal parts of the tissue culture fluid for preparation of the emulsified vaccines.

HUMAN SUBJECTS

In extending to man studies on vaccination performed in laboratory animals, tests on more than a few individuals had to be anticipated. The first persons to participate in these studies were patients paralyzed in recent years by a poliomyelitis infection and who were in residence at the D. T. Watson Home for Crippled Children, Leetsdale, Pa. In addition to patients who had recovered from paralytic poliomyelitis, there were others who were in residence at the Watson Home for such diseases as arthritis, spastic paralysis, and a variety of congenital deformities. After the initial investigations were under way at the Watson Home, additional studies were undertaken at the Polk State School, Polk, Pa.

The numbers of subjects involved in the several experiments under way are indicated in table 3. Of the group of 98 patients at the Watson Home, 51 are participating in studies involving aqueous vaccines inoculated intradermally, and 27 are involved in studies on emulsified vaccines given intramuscularly. All of the 63 individuals at the Polk State School have been given emulsified vaccines intramuscularly.

Because many persons at all age levels have antibody to one or more of the three types of poliomyelitis virus, and because such persons as well as those with no antibody at all have to be used in studies on immunization, it would be well to explain the significance of the presence of serum antibody as an indicator of previous infection.

Immunologic Status of Recently Paralyzed Patients.—Data are presented in table 4 showing the results of immunologic studies in six patients one to two years after their acute paralytic infection. In this group virus had been isolated from fecal samples collected shortly after the onset of paralysis. It is of interest to note that, in the three patients who had poliomyelitis in 1950,
type 3 virus was isolated in two instances, and in both instances antibody for the type 3 virus only was found in the serum two years later. In the one patient from whom type 1 virus was isolated in 1950, antibody for both type 1 and type 3 virus was found in 1952. It is probably correct to conclude that the type 1 antibody resulted from the type 1 infection that occurred in 1950 when the type 1 virus was isolated. It is impossible to say, however, whether or not the type 3 antibody resulted from infection that also occurred in 1950 when virus of this type was in circulation, or at some previous time, or perhaps subsequent to 1950. Of the three patients who were paralyzed in 1951 and from whom the type 1 virus was isolated at that time, antibody for the latter virus only was found in 1952, and there was no demonstrable antibody for either the type 2 or type 3 virus.

The foregoing facts illustrate that the neutralizing antibody response that persists in naturally infected human subjects is type specific, and that it is still present at fairly high levels for at least one or two years after a clinically recognizable infection. It is of further interest that the presence of antibody for one particular type, in a paralyzed person whose serum is devoid of antibody for any other type, probably indicates the immunologic nature of the infecting virus.

Complete data on tests for antibody for the three immunologic types are available in a group of 79 recently paralyzed persons. Table 5 contains a summary indicating (1) the year when paralysis occurred and (2) the antibody status in 1952. Of the 79 patients, 60 have antibody for only one immunologic type; only 19 possess antibody for more than one type.”

It would appear from these data that in the area of Western Pennsylvania from which these patients were drawn, the type 3 virus was more prevalent in 1950 and the type 1 virus in 1951, and that all three types were in circulation in 1952. While it is evident that types 1, 2, and 3 viruses have caused paralysis in recent years, it appears that, in the period for which data are available, type 1 has been the most prominent.

Where antibody for more than one type is present, it may be presumed that nonparalytic infection preceded the paralytic attack caused by one or another of the poliomyelitis viruses. Table 6 contains the ages of those with antibody to more than one type, and the immunologic types for which their serum possesses some antibody. As might be expected, the age distribution of those with antibody for more than one type is somewhat higher than in those with antibody for only one type. This is illustrated further in figure 1, which shows not only the age distribution in this particular group of recently paralyzed patients but the frequency with which antibody for one or more than one type was encountered in relation to age.

Poliomyelitis is not a single disease, since it can be caused by any one of three immunologically different viruses. Therefore, persons who have had paralytic poliomyelitis are considered satisfactory for studies on active immunization. From the immunologic viewpoint, these individuals are similar to others without previous history of paralytic poliomyelitis, since there are many persons who may have at one time or another been infected with one or two of the three types of virus without becoming paralyzed. Individuals whose blood is devoid of antibody for any one virus type can not be regarded as immune to the disease because they have not experienced infection with all three types.

Immunologic Status of Subjects Without Prior History of Poliomyelitis.—The age distribution in the 63 subjects at the Polk State School, who are included in the experiments in progress, is shown in table 7. Data available indicate that a high proportion possess antibody for one or more virus types even though these subjects have not had paralytic poliomyelitis. The fact that many more have antibody for more than one type as compared with patients at the Watson Home is probably a reflection of the higher age level in the Polk group. It is of further interest that clinically recognizable poliomyelitis has occurred at the Polk State School only once in the past 35 years, and that was in the late fall of 1951. At that time six cases occurred, one of which terminated fatally. This represented an attack rate of approximately 180 per 100,000. At the time these cases occurred, there was no increased incidence of illness suggestive of nonparalytic poliomyelitis.”

Results of Infectivity Tests in Monkeys: In order that the significance of the foregoing findings may be evaluated in relation to the material inoculated, data are presented in figure 5 showing the infectious activity, for the cynomolgus monkey, of fluids from which the type 2 vaccine was derived. In this figure is indicated the fate of individual animals inoculated intracerebrally with samples of MEF-1 tissue culture pool no. 15, treated with formaldehyde for different intervals. It will be recalled that, on the days indicated, the excess formaldehyde was neutralized with sodium bisulfite. When such fluids were inoculated intracerebrally, not all of the animals could tolerate the concentration of the chemicals contained in these mixtures, and some succumbed shortly after inoculation. In some instances
the symbol for death in the chart also refers to deaths not due to poliomyelitis that occurred before termination of the 30 day observation period. The solid black symbols indicate that paralytic poliomyelitis occurred; the one symbol with the shaded area indicates that no clinical signs of infection were observed in life, but histological evidence of nonparalytic infection was observed when the spinal cords were examined at the end of the 30 day observation period.”

“In concluding these remarks about the experiments on intradermal inoculation with preparations treated with formaldehyde, it is desired to point out that the very distinct rise in antibody that occurred following intradermal inoculation of the type 2 preparation was not paralleled in the subjects similarly inoculated with the types 1 and 3 preparations employed at the same time. The type 1 preparation that had been treated with formaldehyde for 10 days (Mahoney pools 7, 11) exhibited some antigenic activity in many subjects; however, the sérologic tests have not been repeated in all instances and, therefore, are not ready for presentation at this time. There was no indication of any antigenic activity in the type 3 fluid that had been treated with formaldehyde for 10 days. The tentative conclusion that has been drawn is that the effectiveness of the type 2 (MEF-1 pool 15-K) preparation may be related to the presence of a greater concentration of virus in the starting fluid before treatment with formaldehyde, and that this may have been due to the fact that this pool was derived from cultures of kidney rather than testicular tissue. The latter was used in preparing the types 1 (Mahoney 7, 11-T) and 3 (Saukett 10-T) pools, which were the only ones available at the time these experiments were undertaken. Further study will indicate whether these differences are related to concentration of virus in the respective pools, or to qualitative differences among strains with respect to their sensitivity to formaldehyde.”

In the first study carried out at the Polk State School (experiment P-l) a group of 16 subjects were inoculated with adjuvant vaccine no. 2, the components of which were as follows: for type 1, Mahoney pool 7, 11-T treated with formaldehyde for 7 days; for type 2. MEF-1 pool 15-K treated with formaldehyde for 10 days; and for type 3, Saukett pool 23-K treated with formaldehyde for 7 days.

Each tissue culture fluid was emulsified separately with an equal quantity of oil and was pretested for sterility before the three types were blended by mixing 4 ml. of each together with 8 ml. of mineral oil to disperse the rather thick emulsion. A dose of 1 ml. of this mixture was given intramuscularly; it contained 0.1 ml. of the formaldehyde-treated tissue culture fluid for each type, and the total quantity of tissue culture fluid in the 1 ml. dose was 0.3 ml.”

It is of interest to note in figure 12 that, in recently paralyzed persons, most of whom were children, a high proportion possess antibody for the type 1 virus; and it appears that when antibody is present it is usually found in high concentration. In the group of persons without a prior history of poliomyelitis, some had no demonstrable antibody while others had antibody that was readily measurable; a point of particular interest is that, in general, the level of antibody is lower than in those who were recently paralyzed. The most reasonable explanation for this difference is that a shorter interval of time had elapsed between infection and testing in the group of recently paralyzed patients, most of whom were children, as compared with the group who had not been paralyzed, and therefore, in whom the time of infection could not be dated.

It is interesting to observe that the 15 subjects in experiment P-l have essentially the same distribution of antibody as the group of persons without prior history of poliomyelitis, from which they were drawn, except, of course, for the fact that there are not, in the vaccinated group, any who had no demonstrable antibody before vaccination. Following vaccination it appears that the distribution of antibody titers compares well with those of recently paralyzed patients.”

“Of the 16 individuals for whom data are available in experiment P-l, it is clear that they exhibit essentially the same distribution of prevaccination antibody levels as is found in the larger group of persons without a prior history of poliomyelitis, and that after vaccination there is a wide scatter, with a substantial number now with antibody levels comparable to that found in recently paralyzed patients.”

“Repeatability of Experiments.—The significance of the observations described in this report will not be realized until it can be demonstrated that the effects produced are repeatable. Sufficient time has not elapsed to allow the completion of all repeat experiments now in progress.”

“Comparative data on the antibody-inducing effect in humans of poliomyelitis virus vaccines with and without emulsification with mineral oil are meager. However, the trend in the observations here reported is in keeping with the results of studies with influenza virus vaccines, in which it has been found that a concentration of virus in sodium chloride solution, just below the threshold necessary to induce antibody formation, can be rendered antigenically effective if prepared in a water-in-oil emulsion. The efficacy of this method for inducing antibody formation with small quantities of antigen has been discussed elsewhere. When a sufficient concentration of influenza virus is emulsified with mineral oil and inoculated into experimental animals, it has been possible to evoke the formation of much greater concentrations of antibody than develop as a result of experimentally induced infection. Similar observations are being accumulated in studies with the poliomyelitis virus.”

“All of the subjects included in the experiments here described possessed, prior to inoculation, antibody for one or another of the virus types. This is emphasized since it is conceivable that the immunologic response of such individuals even to virus for which they possess no detectable antibody may be different from that of persons who have no antibody for any type and are first brought into contact with the poliomyelitis virus when inoculated with vaccine. The answer to this question will soon be forthcoming.”

“There are some who may object to the use of kidney tissue as the source of virus because of the unanswered question regarding organ damage by immunization with material emulsified with mineral oil. Although many theoretical arguments as well as examples by analogy may be brought forth to indicate that such effects are improbable with the material employed, it still remains for studies that are underway to indicate whether or not it will be necessary to purify the virus by chemical or physical means in order to insure that such immunologic reactions will not occur if such vaccines are used on a large scale or if more than one injection has to be given.”

SUMMARY AND CONCLUSIONS

“Preliminary results of studies in human subjects inoculated with different experimental poliomyelitis vaccines are here reported. For preparation of these vaccines virus of each of the three immunologic types was produced in cultures of monkey testicular tissue or
monkey kidney tissue. Before human subjects were inoculated, the virus was rendered noninfectious for the monkey by treatment with formaldehyde.”

Although the results obtained in these studies can be regarded as encouraging, they should not be interpreted to indicate that a practical vaccine is now at hand. However, it does appear that at least one course of further investigation is clear. It will now be necessary to establish precisely the limits within which the effects here described can be reproduced with certainty.

Because of the great importance of safety factors in studies of this kind, it must be remembered that considerable time is required for the preparation and study of each new batch of experimental vaccine before human inoculations can be considered. It is this consideration, above all else, that imposes a limitation in the speed with which this work can be extended. Within these intractable limits every effort is being made to acquire the necessary information that will permit the logical progression of these studies into larger numbers of individuals in specially selected groups.”

doi:10.1001/jama.1953.13.1081

In Summary:

• It was not until the report of Burnet and Macnamara in 1931 that there was suspicion of existence of more than one immunologic variety (i.e. strain) of Polio
• The first attempts at immunization of man were made by Brodie and Kolmer in the early 1930’s but these were discontinued because the preparations employed were found unsafe for human use
• Salk claims that this failure may have been due to there being more than one strain
• The existence of at least two distinct types was reported by Kessel and Pait in 1948 and by Bodian, Morgan, and Howe jn 1949
• Soon both groups of investigators reported the existence of a third immunologic variety
• The Committee on Typing of the National Foundation for Infantile Paralysis, through the cooperative effort of investigators at four universities, started a study to determine whether only three types exist
• On the basis of their findings of the first 100 strains (i.e variant that possesses unique and stable phenotypic characteristics) examined, they concluded that vaccination attempts only needed to be concerned with the three varieties of the “virus” that had been found
• These “strains” are referred to as type 1 for those that are related to the Brunhilde prototype; as type 2 for those that resemble the Lansing strain; and as type 3 for those that resemble the Leon strain
• The tissue culture technique by Enders allowed the “virus” to be propagated in cultures of non-neural tissue which was used as a new source for creating more “virus”
• Salk claimed that Enders discovery supported the hypothesis that the “virus” may multiply in non-neural tissue and only then be disseminated from the portal of entry or site of multiplication to the central nervous system
• This hypothesis suggested further that the paralytic sequel of infection might be avoided if an antibody barrier could be created in the blood stream to intercept the “virus” before invasion of the central nervous system (using one unproven theory to prop up another)
• Another significant observation was the finding of “virus” in the blood stream prior to the onset of paralysis both in experimental animals and in man
• This finding of “virus” in the blood by indirect measures (as no “virus” was never purified/isolated) added support to the hypothesis that the “viruses” (i.e. more than one) of poliomyelitis are not strictly neuronotropic
• Further indication that viremia (“virus” in the blood) may represent a stage in the pathogenesis of central nervous system involvement in humans was presented by Hammon
• An observation by Sabin on the relationship of age of mice to the development of paralysis provided the basis for an attractive hypothesis to explain why crippling occurs less frequently and less severely in younger persons
• Salk claims the reasonable immunologic explanation (based on nothing but hypotheses) for the once mysterious epdemiological behavior of poliomyelitis was now well established
• Systematic investigations on the “natural” activity of the “virus” could now be carried out through the application of Enders’ tissue culture technique not only for the isolation of “virus” (meaning it still had yet to be isolated) but also for the quantitative estimation of antibody for all three immunologic types
• Brodie and Kolmer in the early 1930’s attempted immunizations of Polio
• According to Salk, their studies failed as they were conducted:
  1. before precise knowledge regarding pathogenesis and immunologic complexity were available
  2. before there had been accumulated experience with the many strains that possess different pathogenic characteristics
  3. before methods had been developed for adequate purification of “virus” from suspensions of central nervous system tissue
  4. before the dynamics of “virus” inactivation or modification were  well enough understood
• In other words, Brodie and Kolmer had no clue what they were doing nor what they were working with
• More than a decade later, Morgan demonstrated that a formaldehyde-treated suspension of central nervous system tissue from monkeys containing a type 2 strain of poliomyelitis “virus” did, after a rather rigorous schedule of immunization, induce the formation of appreciable quantities of antibody (i.e. non-specific theoretical antibody measurements meant success)
• Another approach that was suggested for inducing immunity was that of the feeding of attenuated living “virus”
• Koprowski reported the results of studies showing an antibody response in 20 humans fed a preparation of type 2 “virus,” which had been propagated in the central nervous system of rodents
• Salk posed 3 questions for inducing immunity:
  1. A rich source of “virus” reasonably free of extraneous antigenic material (i.e. not purified)
  2. A method for destroying pathogenicity without completely destroying antigenic capacity
  3. A means for enhancing antigenic activity if the richest source of “virus” available after treatment for destroying pathogenicity proves to be inadequate or borderline in its effectiveness
• “Virus” of the three immunologic types used in these studies were derived from cultures of monkey testicular and monkey kidney tissue
• The destruction of infectivity was accomplished by treatment with formaldehyde
• In some experiments emulsification with mineral oil employing the technique of Freund was used for enhancing antigenic activity
• Salk suggested the possibility that invasion of the central nervous system by certain strains that occur in nature at different times may not be via the blood stream but by direct extension along nerve pathways
• The acceptance of the idea that invasion of the central nervous system may be primarily via the blood stream rather than primarily along neural pathways had increased the likelihood of achieving a vaccine and lowered the requirements for doing so
• The vaccines employed in these studies were prepared from “virus” propagated in roller tube m cultures of two different tissues derived from monkeys
• Monkey rather than human tissue was decided on for producing the “virus” for these experiments in human subjects because of the possibility that human tissue might be contaminated other “viruses”
• Salk states the plan was to use formaldehyde to kill the Polio “virus” in human tissue cultures but he was unsure it would destroy other agents of human disease which may be in the sample (i.e. not purified/isolated)
• Testicular tissue was the first monkey tissue used for culturing the “virus,” yet they decided to investigate other monkey tissues for comparison
• Enders and associates had employed human sources such as the foreskin, embryonic skin and muscle, the kidney, and the uterus
• The advantage of the testicular cultures in yielding “virus” for long periods is offset by the greater abundance of “virus” in kidney cultures in the early days after “virus” inoculation (meaning kidney cultures were more toxic eatly) so Sabin decided to use monkey kidney cells
• Mixture no. 199 was used as a medium and consisted of:
  1. A large number of amino acids
  2. Certain nucleic acid constituents
  3. Vitamins
  4. Minerals
  5. Other essential growth-promoting or growth-sustaining substances
• The roller cultures in which “virus” was propagated from monkey kidneys contained approximately 100 mg. of minced tissue with 4 ml. of nutrient fluid
• Strains for the vaccine were selected quite arbitrarily (i.e.randomly) and principally on the basis of the regularity with which they could be propagated in relatively high titer in tissue culture
• 3 strains were used for this study:
  1. The Mahoney strain was adapted to tissue culture from monkey central nervous system tissue
  2. The MEF-1 strain was transferred to tissue culture from the spinal cord of paralyzed mice
  3. The Saukett strain was “isolated” in this laboratory by direct inoculation of tissue culture with a fecal specimen from a patient paralyzed in 1950
• The Mahoney strain was considered highly pathogenic by any route, whereas the MEF-1 and Saukett strains are essentially nonpathogenic except when given intracerebrally
• When undiluted tissue culture fluids containing high concentrations of either of the latter two are injected by nonneural routes, paralysis will occur infrequently
• In other words, the “strains” are created from different methods and combinations of tissue culture/fluids where some preparations are more toxic than others
• It was the consensus that tests for safety in humans should include the intracerebral inoculation of monkeys
• In all of the material employed for human beings, the absence of infectivity for the cynomolgus monkey was established in prior tests by the intracerebral inoculation of 0.5 ml. of fluid in 6 to 10 animals
• Salk decided that the fact that amounts of “virus” that produce paralysis when given intracerebrally are not paralytogenic when given intramuscularly or intravenously suggests that the intracerebral safety test may provide a more than adequate margin in certifying a given preparation as safe for use in human subjects
• In other words, if a monkey did not become paralyzed after having the toxic tissue cultured goo injected into their brains, the vaccine was “safe” for humans
• The mineral oil used in these experiments was known as “drakeol no. 6,”
• The preparation of purified emulsifying agent was mixed as a 3% solution in the mineral oil and sterilized by Seitz filtration which was then mixed with equal parts of the tissue culture fluid for preparation of the emulsified vaccines
• The first persons to participate in these studies were patients paralyzed in recent years by a poliomyelitis infection and who were in residence at the D. T. Watson Home for Crippled Children, Leetsdale, Pa.
• In addition to patients who had recovered from paralytic poliomyelitis, there were others who were in residence at the Watson Home for such diseases as arthritis, spastic paralysis, and a variety of congenital deformities (i.e. their initial test subjects were not healthy)
• Of the group of 98 patients at the Watson Home, 51 are participating in studies involving aqueous vaccines inoculated intradermally, and 27 are involved in studies on emulsified vaccines given intramuscularly
• All of the 63 individuals at the Polk State School have been given emulsified vaccines intramuscularly
• Salk admits many people at all age levels have antibody to one or more of the three types of poliomyelitis “virus” (so much for antibody specificity)
• Those who have antibodies were used along with those who have no antibody at all in the studies on immunization but it was assumed that those with antibodies had prior infections
• In one of three patients from whom type 1 “virus” was isolated in 1950, antibody for both type 1 and type 3 “virus” was found in 1952
• Salk states that it is probably correct to conclude that the type 1 antibody resulted from the type 1 infection that occurred in 1950 when the type 1 “virus” was “isolated”
• However, he admits it is impossible to say whether or not the type 3 antibody resulted from infection that also occurred in 1950 or at some previous time or perhaps subsequent to 1950 (i.e he is assuming it was due to a previous infection rather than this result confirming that antibodies are non-specific)
• Salk claims that the presence of antibody for one particular type, in a paralyzed person whose serum is devoid of antibody for any other type, probably indicates the immunologic nature of the infecting “virus“
• 19 of 76 possessed antibody for more than one type
• Where antibody for more than one type is present, it may be presumed that nonparalytic infection preceded the paralytic attack caused by one or another of the poliomyelitis “viruses”
• Salk states that Polio is not a single disease, since it can be caused by any one of three immunologically different “viruses”
• He then offers up an excuse for why using unhealthy paralyzed patients for his study is the same as using healthy subjects:
  1. From the immunologic viewpoint, these individuals are similar to others without previous history of paralytic poliomyelitis, since there are many persons who may have at one time or another been infected with one or two of the three types of “virus” without becoming paralyzed
  2. Individuals whose blood is devoid of antibody for any one “virus” type can not be regarded as immune to the disease because they have not experienced infection with all three types (a huge unprovable assumption)
• Data available indicated that a high proportion possess antibody for one or more “virus” types even though these subjects have not had paralytic poliomyelitis
• Oddly, Salk claims that the fact that many more have antibody for more than one type as compared with patients at the Watson Home is probably a reflection of the higher age level in the Polk group (which he is assuming because they are older, they encountered the “viruses” in the past even though they had no history of Polio)
• Individual animals were inoculated intracerebrally with samples of MEF-1 tissue culture pool no. 15, treated with formaldehyde for different intervals
• When such fluids were inoculated intracerebrally, not all of the animals could tolerate the concentration of the chemicals contained in these mixtures, and some succumbed shortly after inoculation
• Salk noted that the very distinct rise in antibody that occurred following intradermal inoculation of the type 2 preparation was not paralleled in the subjects similarly inoculated with the types 1 and 3 preparations employed at the same time
• There was no indication of any antigenic activity in the type 3 fluid that had been treated with formaldehyde for 10 days
• The tentative conclusion that has been drawn is that the effectiveness of the type 2 (MEF-1 pool 15-K) preparation may be related to the presence of a greater concentration of “virus” in the starting fluid before treatment with formaldehyde, and that this may have been due to the fact that this pool was derived from cultures of kidney rather than testicular tissue
• In other words, the response generated is directly related to the tissues and chemicals used for culturing
• In the first study carried out at the Polk State School (experiment P-l) a group of 16 subjects were inoculated with adjuvant vaccine no. 2, the components of which were as follows:
  1. For type 1, Mahoney pool 7, 11-T treated with formaldehyde for 7 days
  2. For type 2. MEF-1 pool 15-K treated with formaldehyde for 10 days
  3. For type 3, Saukett pool 23-K treated with formaldehyde for 7 days
• Each tissue culture fluid was emulsified separately with an equal quantity of oil and was pretested for sterility before the three types were blended by mixing 4 ml. of each together with 8 ml. of mineral oil to disperse the rather thick emulsion
• In the group of persons without a prior history of poliomyelitis, some had no demonstrable antibody while others had antibody that was readily measurable
• The 15 subjects in vaccine experiment P-l had essentially the same distribution of antibody as the group of persons without prior history of poliomyelitis
• There were none in the vaccinated group who had no demonstrable antibody before vaccination
• Salk states that the significance of the observations described in this report will not be realized until it can be demonstrated that the effects produced are repeatable yet sufficient time had not elapsed to allow the completion of all repeat experiments in progress
• Comparative data on the antibody-inducing effect in humans of poliomyelitis “virus” vaccines with and without emulsification with mineral oil are meager
• When a sufficient concentration of influenza “virus” is emulsified with mineral oil and inoculated into experimental animals, it’s possible to evoke the formation of much greater concentrations of antibody than develop as a result of experimentally induced infection (which logically shows it is the mineral oil causing a spike in antibodies, not the “virus”)
• Salk again states that all of the subjects included in the experiments here described possessed, prior to inoculation, antibody for one or another of the “virus” types
• This is emphasized since it is conceivable that the immunologic response of such individuals even to “virus” for which they possess no detectable antibody may be different from that of persons who have no antibody for any type and are first brought into contact with the Polio “virus” when inoculated with vaccine
• Salk states there are some who may object to the use of kidney tissue as the source of “virus” because of the unanswered question regarding organ damage by immunization with material emulsified with mineral oil
• It still remained for studies that are underway to indicate whether or not it would be necessary to purify the “virus” by chemical or physical means in order to insure that such immunologic reactions will not occur if such vaccines are used on a large scale or if more than one injection has to be given
• Salk warns that although the results obtained in these studies can be regarded as encouraging, they should not be interpreted to indicate that a practical vaccine was at hand
• Because of the great importance of safety factors in studies of this kind, it must be remembered that considerable time is required for the preparation and study of each new batch of experimental vaccine before human inoculations can be considered
• Salk concludes that it is this consideration, above all else, that imposes a limitation in the speed with which his work can be extended

It is pretty astounding the logical leaps that occur in Salk’s paper. From equating the lack of paralysis in intracerebrally injected monkeys to safety in human inoculations to assuming previous Polio infection for those who had antibodies to one or more strain of Polio even though they had no history with the disease, it is clear Salk was viewing his data with a predetermined bias. He was determined to make his data fit his viewpoints so that he could achieve the desired favorable conclusions in order to introduce a vaccine. Even with his tempering of expectations and his plea for time, Salk’s vaccine, while hailed as a savior by the pharmaceutically-controlled MSM, was nothing but a disaster for those injected with it. Upon reading the illogical conclusions and evidence presented in Salk’s paper, there shouldn’t be any mystery as to why his vaccine was such a spectacular failure.

Interestingly, at the end of the study was this eerily prescient warning about the future of medicine:

“Laboratory Medicine.—In most branches of medicine the individual physician is still the indispensable man and his functions cannot as yet be mechanized. In the field of laboratory medicine, however, there is an increasing tendency to relegate to a secondary position the role of the individual and to exalt the apparatus, the test, and the report. This accentuation of the inanimate in medicine is a trend to be deplored and strongly resisted. Like all habits it reduces the resourcefulness of the individual, and it is especially pernicious in that it further weakens those who are already irresolute.

There are physicians in all branches of medicine who at times are unable to reach a prompt diagnosis on clinical grounds alone. Before the era of the ascendancy of laboratory medicine such men would often call on a more experienced colleague for consultation. In most cases such tutorial help would elucidate the clinical features present and would leave the physician much richer in knowledge not only for the benefit of the patient at hand but also for the future. With the rise of laboratory medicine the role of the clinical consultant has been to a large extent erased, and physicians have tended more and more to rely on the laboratory to make the diagnosis for them. Unfortunately, those physicians whose clinical judgment is the poorest are also the ones who are most inclined to accept laboratory reports without question as being final and impeccable. Such a blind dependence on laboratory diagnosis does very little to develop clinical judgment and skill.“

—Marcus Rayner Caro, M.D., Diagnostic Pitfalls of Dermal Pathology, A. M. A. Archives of Dermatology and Syphilology, January, 1953.