Popular Science Monthly/Volume 57/June 1900/Preventive Inoculation I
|PREVENTIVE INOCULATION. (I.)|
By Dr. W. M. HAFFKINE.
DIRECTOR-IN-CHIEF, GOVERNMENT PLAGUE RESEARCH LABORATORY, BOMBAY.
IT was due to certain particularly favorable circumstances that the first ideas on preventive inoculation were gathered from observations on smallpox patients. Such circumstances were presumably the following:
a. It is a disease which attacks epidemically, in a short time and within a small area, large numbers of people, thus permitting of easy comparisons and suggesting conclusions from the facts observed.
b. Its fatality is comparatively small, so that after each outbreak a large number of convalescent persons remain alive to serve as objects for future observation and comparison.
c. These convalescents are marked and are thus easily distinguishable from the rest of the population who have not been attacked, and even the severity of the disease they have gone through is, so to say, written down on their faces and bodies.
d. The disease is easily communicable, owing to the infectious matter appearing on the surface of the patient's body in the pustules.
It was easy, therefore, to notice in this case, as was indeed very early done in the East, that a person who has gone through one attack, as shown by his pitted face, very rarely suffers even during severe subsequent epidemics. Smallpox, like other epidemic diseases, breaks out in some years in very fatal, in others in milder forms. It is admissible that by a mixed process of thought and faith an impression insensibly gained ground that it was lucky to have been touched by the smallpox deity—of course, not in years when that deity appeared in terrifying mortality. Accordingly, in times of mild outbreaks people would not be very careful in avoiding infected persons, and would even seek their company so as to get infected from them. The practice of intentionally rubbing one's skin with a pustule, or with bits of it, from an attacked person, must have been a subsequent stage.
Such or a similarly gradual development of ideas may explain why it is impossible to fix a date or place for this discovery, which indeed goes back to the darkness of antiquity. Research points to its practice among the Chinese and Hindus in very ancient times. The Chinamen induced a mild attack by inserting a crust from a smallpox pustule into the nostrils. The Hindus, on the contrary, used the fluid pus, which they inoculated under the skin of the arm. In either case, in the course of a week, the inoculated was attacked by some slight preliminary symptoms followed by an eruption, sometimes profuse, sometimes scanty, and then the disease would run its ordinary course. The only difference between an attack caused by inoculation and that caused by natural infection was, as a rule, the milder nature of the former, especially when the matter for inoculation was taken from a notoriously mild case. The result, however, was by no means certain. A mild form of an infectious disease may be due either to the virus being of a weak nature; and then such a virus would be the desired one for inoculating persons seeking artificial protection; or else the mildness of the case may be due to the patient himself being of a resistant organization, in which case, though exhibiting mild symptoms himself, he may be harboring an intense form of contagion, apt to cause a severe outbreak when transferred to other less resistant persons. Many plans were consequently adopted to secure with more certainty a mild artificial infection. Some of these were directed to the treatment of the patient preparatory to inoculation, others to the preparation of the infectious matter in order to attenuate its virulence. The Brahmans, who were the operators in India, in addition to selecting material from patients with a mild form of the disease, were accustomed not to employ the pus at once, but to keep it wrapped up in cotton wool for a period of about twelve months, and thus to weaken its power. They inoculated in the early part of the year, at the time when smallpox prevailed, and the practice they used was to moisten with water a bit of cotton wool prepared in the previous outbreak, to place it on the arm of the person to be inoculated, and to prick the arm, through the wool, over an area of about the size of a twenty-five cent piece. In a few days a vesicle would appear at the seat of the inoculation, which later on developed into a pustule and eruption. Notwithstanding these precautions, great variation in the results was observed, and many succumbed to the operation; but those that passed through it safely were proof against further attacks.
Besides the personal risk to the inoculated, the illness produced in them was infectious to others, and unprotected persons coming in contact with the inoculated were likely to get infected from them. The latter result was largely avoided by the practice adopted by the Brahmans of inoculating all the inhabitants of a family or village at the same time. The benefits secured under the above precautions were considered far to outweigh the risks of inoculation.
With the extension of smallpox westward the system of artificial protection spread toward Europe through the intermediary of travelers and merchants. The Arabs and Turks appreciated its benefits at an early date. The slave dealers supplying the bazaars and harems of Constantinople adopted the system to protect against disfigurement their Circassian and other live stock. In the early part of the eighteenth century the method was made known to the English practitioners by Lady Mary Wortley Montagu, the wife of the English ambassador at Constantinople, who had her two children inoculated according to the Turkish system. Curiously enough, it was soon afterward discovered that a similar method was in practice among the peasants of some of the districts in Wales and the Highlands of Scotland, and had long been known there as 'buying the smallpox.' When inoculation was given a more extensive trial it was found, in England as in the East, that the effect of it was decidedly beneficial, but fraught with danger. At first one in every fifty of those operated upon succumbed to the consequences of inoculation. By improved methods the mortality was gradually reduced to one in a thousand; but the most serious danger lay in the spread of infection to healthy persons. The precaution of inoculating whole groups of inhabitants at one time, or of keeping the inoculated apart from the healthy, as had been practiced by the Brahmans ages ago, was overlooked, and the result was often disastrous to the community.
It was at this time that Jenner achieved great progress and threw a vast amount of new light on the question. As is well known, he started from a belief that existed in the west of England, that cowpox was a bovine form of smallpox, and that the milkers who attended on cows suffering from that disease and who became infected with the eruptions on the teats and udders, passed through a mild illness, which rendered them immune against smallpox. Jenner determined to put this tradition to the test, and succeeded in establishing, by a few accurate and well-planned experiments, a series of most important facts.
He showed, first, that cowpox could be artificially given to the cow by infecting it with virus from a smallpox patient, and that the disease thus produced was transferable by inoculation from cow to cow.
He showed further, that by having been bred in the tissues of the cow, the virus lost its intense infective properties for man. When the matter from an artificially infected animal was transferred by inoculation to a human being, it produced at the seat of its insertion a discrete vesicle, which was not followed by a general eruption, as would often be the case with the original smallpox virus.
Though the illness thus induced was not infectious in the sense that it would not be communicated spontaneously from person to person, it could be so transferred artificially by inoculating patients with the lymph from a ripe human vesicle.
When transferred from cow to cow or from man to man the matter preserved unchanged the same property of producing the mild inoculation vesicle, harmless to the patient and to his surroundings; and thus a matter for inoculation was obtained of invariable strength, what was called later on, by Pasteur, 'virus fixe.'
The last and the most essential property which Jenner demonstrated to belong to the substance in question was the following: A man who had been inoculated with that substance could afterward be with impunity infected with a virus taken direct from a smallpox patient; the inoculation would be either abortive altogether or the effect much milder than in a man not so prepared. Jenner concluded from this most striking result that the inoculation with the matter cultivated by him in the cow would protect a man forever against contamination with smallpox, and he called that matter 'vaccine/ or cow lymph.
Jenner's experiments produced an immense impression throughout the world, and inoculation according to his system, which was called 'vaccination/ was rapidly applied to large numbers of people. When outbreaks of smallpox occurred in the midst of vaccinated communities, observations began to come in as to the actual effectiveness of the method in protecting against the disease.
These observations proved that the system possessed an undoubted and exceedingly high beneficial effect, though the following two restrictions had to be imposed upon the originally conceived expectations:
1. The protection was not absolute. In every outbreak of smallpox a number of patients were and are met with who are attacked, generally mildly, but also in some cases fatally, though they had undergone a successful vaccination, some even at a comparatively recent date before the attack. Only the proportion of such patients to the whole of the vaccinated community is very markedly smaller than the proportion of attacks in the non-vaccinated; and also the severity of the attack, as well as the proportion of deaths to attacks, is in the vaccinated much smaller.
2. This favorable difference between the outbreaks among vaccinated and non-vaccinated is maintained not for life, but for a limited number of years, and disappears gradually, and at length altogether, unless the individuals be revaccinated. Observation has shown that the period during which the protective effect of vaccination lasts extends over from three to seven years.
Vaccination very rapidly displaced inoculation, and spread to every part of the civilized world. The results have been dwelt upon in innumerable books and pamphlets. At present great outbreaks of smallpox have become very rare, at least in the civilized part of the world, and there is a tendency to forget or ignore the devastations they used to cause.
The first successful attempt in extending the system of inoculation to other diseases was made only after the discovery of the fact that 'infection' is caused by living animal or vegetable parasites, capable in the majority of cases of being cultivated and bred in artificial media outside the animal body. Pasteur found that he was able to effect protection against disease similar to vaccination against smallpox by the use of such artificially bred micro-organisms.
It may be interesting to relate that this important discovery was made unintentionally, and represents one of those happy 'accidents' which occur to those who diligently search. Pasteur had been working with cultures of chicken-cholera microbes, an extremely fatal form of virus when it is introduced into fowls and small birds. It so happened that one of his cultures was left forgotten in the incubator when work was stopped for the vacation. On the return of Pasteur and his assistants the experiments were continued. When the bottle was discovered, thinking that the microbes might have been exhausted or dead from long starvation, Pasteur tried to make what is called a fresh culture of them, by inseminating a sample from the old bottle into a freshly prepared nutritious broth. The microbes were not dead, and multiplied and grew luxuriantly; but when they were injected into a fowl they caused only a transient and non-fatal disease. To make a fresh start, Pasteur took some old blood, which he had drawn a long time previously from a chicken-cholera fowl and preserved in a cupboard in the laboratory in a sealed-up tube, and made a culture with the material that was in that tube. The culture thus obtained killed fresh fowls as usual, but when it was injected into the bird that had resisted the first culture it resisted this injection also. Pasteur, who excelled all men I ever knew in his ability of quickly analyzing and discerning true connections between facts, required no further hints. Others might perhaps have dwelt on the peculiarity of the fowl that happened to resist the injections, or on some other circumstances. Pasteur relinquished this and other suggestions at once, and thought of the microbe. The fact that old specimens of microbes may become impotent when injected into animals was known to him, and was readily explained by the vitality of such microbes being lowered or exhausted by starvation. But, then, such a microbe when transferred into a fresh medium, if not dead, generally regains its vigor, and after that, when inoculated into an animal, it produces its usual effect. The remarkable circumstance about the culture left in the incubator was that even when it was transferred into a fresh medium and its vitality renewed, it remained still impotent. Pasteur concluded from this that an infectious microbe possesses two distinct properties: one, which it shares with.any other living being—viz., vitality—which may be weakened or strengthened according to the conditions of life and food; and another, which he considered as its 'virulence/ its power of causing diseases, which may be also weakened or strengthened by special means, but which is quite independent of 'vitality.'
The lucidity of thought of which Pasteur made proof on this occasion was magnificent. Later researches confirmed and explained these facts with a singular completeness, and now the idea, as is always the case, looks simple and self-evident. One must remember that at that time Pasteur had every reason to believe that disease is caused by the mere fact of a foreign micro-organism of a given species penetrating and settling down to live in the system of a man or animal. Its capability of living there, i. e., its vital properties, seemed all that was necessary for causing disease. It was only later that it was found that pathogenic microbes cause diseases by producing so-called toxines or poisonous substances distinct from their own bodies and separable from them. The process may be illustrated by a comparison, for instance, with a cobra or any other animal producing a special venom. By starvation or some other treatment the vitality of the cobra may be temporarily weakened. When it obtains fresh food again and gets generally in good condition, it recovers, without its ability of producing venom having been in any way impaired. On the other hand, a snake may be by an operation deprived of its fangs and power of secreting poison without its health and strength being in the least affected. Pasteur at once asserted that in a similar way it was possible by starvation to weaken a breed of microbes without their virulence being diminished, and, on the other hand, to deprive them of their power of producing disease without impairing their vitality, though what the above power consisted in he did not know. He called the latter result attenuation of a virus. An attenuated virus in his meaning is therefore a special breed of pathogenic microbes which can be maintained, by suitable breeding, in best conditions of health, but which has lost either partially or entirely its power of producing poison and disease.
Pasteur extracted from the few experiments related above a further most-important conclusion—viz., that such an attenuation was due to and could be produced artificially by the effect of oxidation. This he deduced from the fact that the microbes in the sealed-up tube had not lost their virulence, while those forgotten in the open bottle in the incubator and exposed to the access of air had done so. Oxidation proved indeed to be one of the most general methods of artificially producing attenuated virus, to which method later on were added others—the effect of light, of chemicals, of passage through certain animals, etc.
And, of course, the last and crowning conclusion was that an ordinary, susceptible fowl that has undergone the injection with an attenuated culture becomes immune against a culture which kills other fowls; and that conclusion, in the particular circumstances under which Pasteur was working, proved to be true.
Pursuing the new line of research, Pasteur demonstrated that a protection similar to that obtained against smallpox and chicken cholera could be secured also against anthrax, a disease which, by the destruction it caused among sheep and cattle, was entailing heavy loss on the farmers of France. After a long series of experiments he prepared two specimens of virus, different in strength, but both weaker than the natural contagion, and worked out the proportions in which sheep, horses and cows could be safely injected first with the weakest virus and then with the virus of the somewhat greater strength, after which they became capable of withstanding the strongest anthrax infection.
In honor of Jenner, who was the first to discover the way of preparing a virus of a fixed strength safe to be used for the preventive treatment of men, Pasteur proposed that all such artificially bred, so to say, domesticated forms of microbes be called vaccines, while the word virus be reserved for a contagion growing in nature in a natural condition, or taken direct from an infected individual. The French distinguish between 'vaccin,' which is used as a generic term in Pasteur's sense, and 'vaccine,' which name they reserve for smallpox vaccinia lymph. The word 'vaccination' has been also extended to designate inoculation with artificially vaccinized virus, while the word 'inoculation' is used for the injection of a natural, not vaccinated virus, taken direct from a patient. The latter distinction is, however, not yet strictly maintained in English literature, nor in the subsequent pages of this paper.
Pasteur gave a memorable demonstration of the efficiency of his method of anthrax vaccination. At Pouilly-le-Fort, in the midst of an assemblage of scientists, delegates of agricultural societies, government officials, landlords, farmers and representatives of the press, he performed the following experiment: Sixty sheep were taken; ten of these were put aside, twenty-five were vaccinated with the two attenuated anthrax vaccines at an interval of twelve days, and twenty-five were left untouched. Twelve days afterward the two groups of twenty-five sheep were inoculated with virulent anthrax; and the result was that at the next visit the twenty-five unvaccinated and one vaccinated pregnant female were found dead, while twenty-four out of the twenty-five that had been vaccinated wore perfectly well, and exhibited during the whole time they were kept under observation the same degree of health as the ten sheep that had been put aside for comparison.
An impetus was given by these discoveries to researches having for their object the protection of men against infectious diseases. The most important of these researches was Pasteur's own into the nature of hydrophobia and rabies, and the way of inoculating against that disease. This was followed a few years later by the preparation of a prophylactic against cholera.
Inoculation against hydrophobia was rendered possible by the discovery of the fact that the rabies or hydrophobia virus is found in a pure condition, free from other microbes, in the nervous centers of animals. The material for inoculation is prepared from such nervous matter, the virulence of which is rendered fixe, as will be mentioned below.
The cholera microbe, which was subsequently named comma bacillus, was discovered by Koch in 1883, in the intestinal contents of cholera patients. Two years later cholera broke out in Spain, and Dr. James Ferran, a Spanish physician, began inoculating men with living cultures of comma bacillus taken from patients attacked with the disease. The procedure in its essential features corresponded to the pre-Jenner method of inoculation. The failure to fix the strength of the virus used for treatment rendered the method subject to the same uncertainty as that which was connected with inoculation with smallpox virus taken direct from patients. It was impossible to predict the effect of the injections. Comma bacilli taken from cholera patients may, under cultivation, show themselves extremely virulent, or, on the contrary, extremely mild. There are specimens which, when injected into a Guinea pig, even in an insignificant dose, will prove fatal to it, and there are others which will appear harmless when given in a dose seventy times greater. The immediate effect, and the protection caused by the inoculation, must, of course, vary accordingly. The attempt made by Ferran caused great interest, and a number of scientific commissions were sent to Spain from different countries of Europe to study the results of his work. They could, however, come to no conclusion, and the treatment speedily lost its position. Only some seven years later a method was found of fixing the strength of the cholera virus. I was connected with this stage of the work, and it may perhaps present some interest to the reader to relate the way in which the problem was solved, and to show how gradual is the development of ideas by which results in laboratory investigation are arrived at.
It has been mentioned already that the virulence of microbes changes under the influence of different agents in Nature—heat, light, chemicals. When a virus is first obtained from a patient or outside a patient its preceding history, its antecedents, the conditions under which it lived before, are extremely variable. Jenner's method of cultivating the smallpox virus by transferring it from calf to calf secured for that virus uniform conditions of life, and its strength could thus be maintained unchanged for an indefinite length of time. Pasteur, in the preparation of hydrophobia vaccine, followed the same plan, and found in the successive inoculation from rabbit to rabbit a method of propagating the hydrophobia virus in a uniform condition. But attempts made to cultivate in a similar way the comma bacillus by transferring it from animal to animal failed.
The most susceptible animals for the cholera microbe are Guinea pigs. There are two principal methods of ingrafting upon them the virus: Koch's method of administering it through the mouth and leaving it to develop in the intestines of the animal, and Pfeiffer's of injecting it, not into the intestines, but into the abdominal or peritoneal cavity, where the intestines are lodged, by introducing there the virus with a hypodermic needle not allowed to penetrate into the intestines themselves. But by neither of these methods could the microbe be cultivated in an unbroken series of animals, as it became gradually weakened and soon lost its power of affecting such animals. For the purpose in question, cultivation in the peritoneal cavity had the advantage that in a healthy individual the peritoneum is free from other microbes, whereas in the intestines there are always present a large number of micro-organisms which interfere in variable ways with the growth of the particular microbes.
But when one inoculates the peritoneal cavity of a Guinea pig with a dose of cholera microbes sufficient to cause a fatal disease, it is found, when the animal dies, that the microbes have died also. Thus, the attempt to ingraft the virus from a first animal to a subsequent one is checked at the very beginning. This initial difficulty was overcome by merely giving to the first animal a dose larger than was necessary to cause a fatal effect. The animal then succumbs more rapidly, and the microbes have no time to disappear. At the post-mortem examination there is found, in the peritoneal cavity, a small amount of exudate liquid which contains large numbers of those microbes alive. When, however, that exudate is injected into the peritoneal cavity of a second animal that animal does not succumb to the infection, or even if it succumbs one finds that the microbes have again disappeared in this second animal. By starting with % a still larger initial dose one may have three, perhaps four, successive animals affected by the virus, but it invariably ends by being weakened, and finally dies out.
In trying to obviate this result I found, perhaps contrary to expectations, that the exudation liquid should be exposed to the air for a few hours before it is injected into a subsequent animal. This result was contradictory to the effect which Pasteur had found to be exercised by atmospheric oxygen on the virulence of microbes, and it requires at least some provisional explanation. The microbes of cholera differ from a certain number of other microbes in that they stand in need of a free and abundant access of air for growing and multiplying quite satisfactorily. They are deprived of this condition in the peritoneal cavity of an animal. It is possible, therefore, that a certain opposition between the maintenance or development of virulence on the one hand, and a lowering in vitality on the other, takes place while they are cultivated there, and a respite must be given them between each successive 'passage' through the Guinea pig by leaving them for a time in the free atmosphere. Be that explanation true or not, the result is that under such conditions the successive animals inoculated with the virus do succumb, and even in a shorter and shorter time, after the inoculation, the microbe apparently undergoing under such a treatment a progressive increase in virulence. A similar development up to a certain stage was observed by Pasteur when transferring the rabies virus from rabbit to rabbit. The last difficulty that presented itself was the following: The exudation liquid which is found in the peritoneal cavity post mortem varies in quantity; sometimes it is inconveniently large and diluted; sometimes, on the contrary, so scanty that it becomes difficult to collect and transfer it to another animal. I found that this variation stands in connection with the size of the animal, so that a diluted exudation fluid can be concentrated by injecting it into a small animal, while a too much concentrated exudate is rendered more dilute by transferring it to an animal of a larger size.
Thus, by the initial use of more than a fatal dose, by alternating cultivation in an animal with exposure to air, and by attention to the size of the animal employed, a material was obtained which, as mentioned, increased in intensity from the first and proved fatal to animals in a shorter and shorter time after inoculation. Later the virus reached a stage when it killed a Guinea pig of three hundred and fifty grammes weight in eight hours. After that, in each further inoculation the time of eight hours remained stationary, showing that the virus has reached the condition of a 'virus fixe' These experiments were conducted by me in the Pasteur Institute, in 1889 to 1893, simultaneously on the cholera microbe and on the bacillus of typhoid. The two exhibited a number of common features in their nature, and the results as above detailed for the cholera microbe were found valid for the typhoid bacillus also.
Starting from the 'virus fixe' obtained as above, a method of double inoculation was worked out, one with an attenuated virus prepared from the 'virus fixe' and another with the latter itself. The two 'vaccines' when inoculated successively into Guinea pigs, protected them against all possible forms of cholera infection. The vaccines were cultivated on a solid medium, and when the crop of microbes was ready at the end of some twenty-four hours, they were washed off the surface of the medium and used as a kind of medicinal plant. It was found that the substances contained in the microbes preserved to a great extent their immunizing properties even when the microbes were killed by some delicate processes not affecting considerably their chemical constitution. The washings could, therefore, be prepared in dilute solutions of carbolic acid, and employed in the form of preserved vaccines. In 1892 and in the beginning of 1893 I made a series of experiments in Paris, in Netley, in London, in Cambridge, and in Calcutta, with these carbolized cholera vaccines, which had been preserved in sealed tubes for a period of six to seven months, and it was possible to show the protective effect of the method on animals as conclusively as Pasteur had done in the demonstration at Pouilly-le-Fort with anthrax. For the inoculation in man, however, I decided to use at first only unaltered living vaccines, as much more promising than the dead ones, especially from the point of view of the durability of the effect.
[To be concluded.]