by JOAN SCHIAVONE and SYDNEY D. RUBBO Dept. of Bacteriology, University of Melbourne
We are all familiar with the concept of antibodies playing an important role in the protection against infectious disease. Thus a person who has recovered from diphtheria will possess a circulating antibody, usually known as antitoxin, capable of neutralising the diphtheria toxin. This type of acquired natural immunity will, in many instances, protect the individual against recurrence of the same disease. However, in this and many other diseases it is possible to develop specific immune antibodies without exhibit- ing signs of disease. For example, by means of the Schick test it can be shown that many people have protective antitoxin to diphtheria with no history of the clinical disease. If such individuals have not been artificially immunised we can assume they have acquired a natural immunity by sub- clinical infection.
The knowledge that protective antibodies can be induced against many bacterial and viral agents of disease has been utilised in artificial immunisation by the use of vac- cines. A vaccine contains micro-organisms or their toxins which have, by various means, been rendered harmless to the re- cipient and yet which still retain their anti- genicity, i.e., the ability to stimulate the production of antibodies.
The need for a vaccine against poliomyel- itis is apparent when one considers the in- creasing incidence as well as the social and economic implications resulting from the crippling effects of this disease. The diffi- culties inherent in the production of such a vaccine are many because, in this instance, we are dealing with a disease caused by a virus which exists in three immunologically different forms, Types I, II, and III. Thus, the vaccine must contain the three antigenic forms of the virus and each can only be
propagated on living cells. A choice 'be- tween two fundamentally distinct methods of preparing the vaccine is open to the viro- logist. The first is to prepare a killed vac- cine containing the three antigenic types and the second is to make living avirulent virus vaccine of each type. Both methods are being actively investigated and the Salk- type vaccine is an example of the former.
We might briefly compare the Salk-type of a polio vaccine with those which may be available in the future.
1. Salk-type — chemically killed mix- tures of virulent Types I, II, and III.
2. Modified Salk-type — chemically killed mixtures of avirulent Type I and virulent Types II and III.
3. Attenuated type — mixtures of living avirulent Types I, II and II to be given by injection.
4. Attenuated type — individual sus- pensions of avirulent Types I, II and III, each to be given by mouth.
As it is not the object of this present ar- ticle to discuss the relative merits of these possible forms of polio vaccines we will concentrate our remarks on the presently- available Salk-type vaccine.
A significant discovery which was used by Salk and his associates in the develop- ment of his polio vaccine was the observa- tion by Enders in 1949 that the poliomyel- itis virus could be grown in vitro in non- nervous tissue cultures. It was found that a variety of human or monkey tissues, such as kidney and testes, which produce epi- thelial cells, or fibroblasts may be used. The monkey'g kidney has been particularly suc- cessful because it gives a very high yield of epithelial cells which, in turn, give a high virus yield. Over 2,000 tissue cultures may
54 SPECULUM be prepared from a single monkey. Growth
of virus in tissue culture, therefore, made the preparation of a vaccine a practical pos- sibility and the predictably constant growth of the three types of poliomyelitis virus pro- vided the means of producing large quanti- ties of virus under controlled conditions necessary for vaccine manufacture.
The idea of using a formalin-killed virus vaccine was not new. It had been attempted in the early 1930's by Brodie, but at that time there was no convincing evidence of the effectiveness of the formalinised vaccine It was Salk and his fellow workers (which included Dr. P. L. Bazeley, of Melbourne) who elucidated the principles of formalin inactivation of the poliomyelitis virus. The basis of this has been succinctly stated by Salk in an article in the American Journal of Public Health, May, 1954, "As with other antigens, interaction of `formaldehyde with poliomyelitis virus, resulting in de- struction of infectivity, occurs in accordance with the laws that govern a first order che- mical reaction. This means that if conditions are constant throughout the reaction period, the rate, measured on a logarithmic scale, at which virus infectivity is destroyed, is constant. For this reason it is possible to prepare a poliomyelitis vaccine for test that may be said to be free of infectious par- ticles; more than that, it is possible to define the extent of what may be referred to as the "margin of safety" of each batch of such vaccine. Thus the vaccine with which we are concerned is treated in a way that will destroy the infectivity not only of the last virus particle that can be measured, but will destroy the infectivity of many more additional particles that might be postulated to be present and not measurable."
It required further work to establish the correct conditions under which a safe and effective procedure could be adapted to large scale production. It was recognised that the variables which influenced the time required for destruction of infectivity are (1) concentration of free formalin, (2) the temperature at which the reaction is allowed to take place, (3) the pH of the reaction mixture, and (4) the concentration of virus initially present. It was found that, with a constant composition of the medium, free of tissue debris, etc., 1:4,000 formalin solu- tion at a temperature of 36°-37°C. and pH 7 provided suitable constants and the only variable to be determined was the time
taken required for destruction of infectivity.
This was, in the main, dependent upon the concentration of virus in the starting mater- ial and by tissue culture methods an esti- mate of virus concentration was possible with each batch of virus culture. Thus, the fluids harvested from virus infected cultures of each type are tested to establish the time required for inactivation of living virus in that particular batch. This is done by treat- ing with formalin as indicated above and titrating for residual infectivity (by inocu- lation of tissue cultures) on successive days.
The purpose of this test is to ascertain the time required for complete inactivation under the particular conditions that apply in any given laboratory for any given batch, i.e., a new and individual standard is set for each batch. The batch from which the preliminary test is made is then treated for three times the time indicated by the pre- liminary testing. Further titrations for re- sidual infectivity are carried out daily dur- ing the inactivation process. Final tests for safety are made after mixing of all three strains by tissue culture methods and by intracerebral and intramuscular injection of monkeys which are observed clinically and later histologically. Finally, safety tests are made on random samples of the pooled vac- cine from each batch. Thus, each batch of vaccine is tested for infectivity (or safety) three times by tissue culture and animal inoculation over a period of 2-3 months before being issued for use.
A brief survey such as this cannot convey the extent of safety precautions which are carried out in the preparation of the vac- cine, but for those who are interested, a perusal of the paper mentioned above is recommended.
As well as testing for safety the final vaccine is tested for immunological effect- iveness in mice, monkeys and humans. Only fluids having titles of at least 106 units per ml. are used to ensure a sufficient antigenic concentration of virus. The careful control of the inactivation procedure is designed to protect the antigen that is present initially.
. . . . This control is necessary because the formalin used to kill the virus is also cap- able of destroying the viral antigen. How- ever, this is easily controlled as it has been repeatedly demonstrated the time required to significantly reduce the antigenicity is ap- proximately five times that required to ensure absolute inactivation of the virus.
SPECULUM 55
The initial human experiments carried out by Salk et al. showed that antibody to all three types was produced after injection of the killed vaccine and that the levels obtained were comparable to antibody levels reached in the naturally occurring disease. Having satisfied the prerequisites of safety, antigenicity and adaptability to mass production methods it remained only to prove that the observed antibody re- sponses did confer real immunity in man.
Accordingly, a mass trial of the Salk-type vaccine was carried out under the auspices of the National Foundation for Infantile Paralysis in America. The results of the survey and possible inferences to be drawn are contained in the summary report "An Evaluation of the 1954 Poliomyelitis Vac- cine Trials" (American Journal of Public Health, May, 1955), commonly referred to as the Francis Report. Amidst this welter of scientific detail many interesting conclu- sions become apparent. Briefly, they may be summarised as follows:—
(1) There were no significant differ- ences in the incidence of non-para- lytic cases in the vaccinated and ob- served plus placebo groups.
(2) When only cases of paralytic polio- myelitis are considered the vaccine proved 62% -72% effective protec- tion.
Because cases classified as paralytic poliomyelitis might contain cases which were not related to the polio- myelitis virus, analysis was carried out of cases proved by isolation of the virus. Effectiveness was 64% - 84% , and became more clear-cut with the more severe form of polio- myelitis.
(4) Effectiveness of vaccine against the various types were 60% effective against Type I, 70%-80% effective against Types II and III in the 'ob- served' control areas where as in the "placebo" study areas 60% - 70% effectiveness against Type I and 90% or more effectiveness against Types II and III were de- monstrated.
On the basis of these results alone there can be no doubt that the Salk vaccine is a safe and effective weapon against polio- myelitis. However, other facts emerged from this mass testing and also from follow-up work since that time which indicate that
an even higher rate of protection may be expected.
During the vaccine trials it was noted that some batches of vaccine gave uniformly higher rates of protection and also stimu- lated a 'higher antibody response in the re- cipients than others. On this basis the batches were graded as giving "very good, good, fair, poor, trace, or no "antibody response." A grouping of lots observed and estimated gave the following information:—
"Five lots and three combinations have been called good . . . Three lots and two combi- nations are poor, one of them essentially devoid of antigenic activity . . . Two others have little effect for Types I and II, but have fair Type III components. The other lots fall into an intermediate position. It is generally true that the Type I component is less effective than the II and III." When it is realised that the figures quoted in the Francis report include all vaccinated groups irrespective of the effective protective power of the various vaccines it must be admitted that a vaccine which can be consistently graded as good or very good to the three types the protection figure will be significant ly high.
The attainment of good quality vaccine is now being achieved. Experimental inves- tigation of the various batches of vaccine quickly showed that the differences in anti- genicity were due to a destructive effect on the viral antigen of merthiolate, added to the finished vaccine as a preservative. Fur- thermore, this destructive action increased with storage and was most evident with the Type I component. This probably explains the lesser protection against Type I infec- tion revealed in the statistical survey of cases. The question of preservatives has now been largely overcome with resultant improvement in grading of the vaccine.
Improvements have also been made in production which can best be summarised on terms of antibody production in indi- viduals without previous poliomyelitis anti- body. Thus, the 1955 vaccine induced anti- body levels of 1:4 or greater, after the first dose, in approximately 90% or more of groups of individuals. By comparison, 1954 field test vaccines exhibited a somewhat lower antibody response. The geometrical mean leNiels of antibody induced as a result of the first dose of vaccine were 1:8 and 1:16 for 1955 vaccines, and slightly under 1:8 for 1954 preparations.
(3)
The final consideration in gaining maxi- mum antibody response is the finding that the period required for the greatest de- velopment of the hyper-reactive state in Man following primary antigenic stimulus is a matter of months. In the 1954 field trials injections were given at 0, 2, 5 week inter- vals. It has now been shown that this time interval is sufficient to develop the primary response only and that the full potential of the vaccine was not reached in the 1954 trials. Yet the vaccine gave a 64% to 84%
effectiveness in this period. How much more effective the vaccine will be when secondary antibody response is operating, is clear from measurements of antibody levels fol- lowing a booster dose given seven months after the primary vaccination. Antibody levels of 1:8,000 or more are often achieved. For comparison the antibody level resulting from paralytic infection is of the order of 1:1,024.
On this basis then Salk recommends pri- mary intramuscular vaccination of two doses each of 1 ml., separated by a 2-4 week interval with a booster dose not earlier than the seventh month.
The final question to be answered is how long will the effects of the vaccine last.
Broadly, from the evidence of antibody re- sponse to booster injection, it can be pre- dicted that the duration of significant anti- body titres will be maintained for some con- siderable time. Dr. Salk states, "It would appear that if vaccination induces a long lasting alteration in the state of reactivity of the immunological mechanism (as ap- pears to be so) then subsequent contact with the poliomyelitis virus under natural circumstances should cause antibody forma- tion to begin sufficiently rapidly, and might be expected thereby to increase the likeli- hood that long lasting immunity will follow the proper use of a properly-constituted vaccine." In support of this claim Salk gives figures for 27 children vaccinated in the Spring of 1953 or 1954 who, two weeks after their primary vaccination, had anti- 'Iody levels to all types ranging about 1:32.
They maintained good health and were tested for residual antibody level in the winter of the appropriate year. It was found that these children had each experienced natural re-infection as indicated by the sharp rise in antibody level of one type only. Furthermore, the level of the antibody
was higher than that observed in paralytic convalescent.
In conclusion, we might quote from the New York Times, August 28, 1955, com- menting on the vaccine. The Cutter Report which dealt with an investigation of the safety of the vaccine stated what everyone already knew: that some batches of the vac- cine manufactured by the Cutter Labora- tories contained live virus. It did not say what everyone wanted to know: how the virus got there. Since the Cutter incident the standards of safety testing have been increased, as indicated above, so the chances of further accidents are exceedingly slim. The emphasis in America has now shifted from safety to effectiveness. While it is still too soon to reach any conclusions for this year the following figures from Massachusetts are encouraging:
No. children No. Polio Cases per (5-9 years) Cases 10,000 285,000 unvaccinated 429 15
150,000 one shot only 43 2.8 24,000, two or more shots 2 0.8 On these figures it looks as though the Salk Vaccine is living up to the advance an- nouncement it received in April, 1954:
"The vaccine works. It is safe, effective and potent." The decision to manufacture the Salk vaccine in Australia depends on whe- ther the advisers to the Government feel that the safety tests now used are reliable safeguards. Continued experience in Amer- ica will help to crystallise such decisions for or against.
REFERENCES
FERRIS, A. A. (1954), "Immunisation Against Poliomyelitis — The position today," Health Bulletin — J. of Dept. of Health, Victoria, No.
111, 18.
FRANCIS, T., Jr., KORNS, R. F., VOIGHT, R. B., BOISEN, M. HEMPHILL, FAY M. NAPIER, J. A., and TOLCHINSKY, Eva (1955), "An Evaluation of the 1954 Poliomyelitis Vaccine Trials," Am. J. of Pub. Health, Part 11, 45, No. 5.
MELNICK, J. L. (1954), "Application of Tissue Cul- ture Methods to Epidemiological Studies of Poliomyelitis," Am. J. of Pub. Health, 44, No. 5.
571.
SALK, J. E. (1955), "Vaccination Against Paralytic Poliomyelitis — Performance and Prospects,"
Am. J. of Pub. Health, Part I, 45, No. 5, 575.
SALK, J. E. (1954), "Application of Tissue Culture Methods of Epidemiological Studies of Polio- myelitis," Am. J. of Pub. Health, 44, No. 5, 571.
"Symposium on Poliomyelitis" (1953), "Pediatric Clinics of North America, 1, No. 1A (various authors).
SPECULUM. 57
Theatre Night
Early last year the M.S.S. Committee had started thinking about arranging a theatre night to see "Doctor in the House." One of the wide-awake Committee members had heard that the best seller was being filmed Enquiries at the Melbourne distributing agencies were not helpful—they knew noth- ing about it. Some months later, when the film was reviewed in "Punch", the agency concerned conceded that it had been made
— but couldn't say when and where it would be shown in Melbourne. By the end of the year, however, the M.S.S. had suc- ceeded in convincing the Odeon that we wanted a block booking for the first night.
Unfortunately for us, the preceding film was proving a financial success, and our long- awaited first-night was several times post- poned, finally to land during the long vaca- tion. Thus it came about that only the clinical years could be reached and invited to join the fun.
Friday, February 25th, finally arrived.
So did Doctor in the House. So did 500 Medical Students, at the Odeon Theatre.
Ruth was down there early, hoisting the banner of the M.S.S. across the Odeon's en- trance doors. We were pleased to see our banner again — the P.H. lads had borrowed it to decorate Prince Hugo's wedding.
Another stethoscoped character soon ar- rived, dressed in white, except for his black bowler hat. The press photograhpers de- scended on him, and when we last saw him, he was blissfully posing at auscultating a pretty usherette.
Along Bourke St. trotted Melbourne's last hansom horse, drawing Melbourne's last regular hansom cab proudly behind him.
With a flourish of the reins, the driver put on his brakes. Then, the Prince Henry Col- lege of Surgeons dismounted. They had been delivered straight from the Women's Hospital. Clad in Theatre attire, they were dressed for the theatre.
3AW's Radio Roundsman was amusing his radio audience by interviewing the more professional - looking theatre - goers. Those interviewed sang a song or two for the edu- cation of listeners. We fear that portion of the tape recording must have demag- netized itself.
Interval was brightened up for the Anthem. These weje roneoed for the occa- sion, but are now available in more per- manent form in that magnificent collection
— The Songbook.
Then came Doctor in the House. Here we felt quite at home. We had all been told
"the traditions of this hospital . . . " Who hadn't stood at the door of the theatre and been told: "You don't learn Surgery from the doorway!" Hadn't we seen patients told that the greatest danger in their opera- tion was Blood!
Then it dawned on us, Doctor in the House had been filmed in Melbourne, with a story founded on fact. Box-office meant it had to be watered down for the English public to accept.
—R.G.