It has been mentioned that Metchnikoff, in a publication in 1883, attempted to explain immunity on a purely cellular basis. It has been known since Haeckel’s first observation in 1858 that certain of the white corpuscles do engulf solid particles that may get into the body, and among them bacteria. Metchnikoff at first thought that this engulfing and subsequent intracellular digestion of the microörganisms were sufficient to protect the body from infection. The later discoveries (discussed in considering Ehrlich’s theory of immunity) of substances present in the blood serum and even in the blood plasma which either destroy the bacteria or neutralize their action have caused Metchnikoff to modify his theory to a great extent. He admitted the presence of these substances, though giving them other names, but ascribed their formation to the phagocytes or to the same organs which form the leukocytes—lymphoid tissue generally, bone marrow. It is not within the province of this work to attempt to reconcile these theories, but it may be well to point out that Ehrlich’s theory is one of chemical substances and that the origin of these substances is not an essential part of the theory, so that the two theories, except in some minor details, are not necessarily mutually exclusive.
Sir A. E. Wright and Douglas, in 1903, showed that even in those instances where immunity depends on phagocytosis, as it certainly does in many cases, the phagocytes are more or less inactive unless they are aided by chemical substances present in the blood. These substances act on the bacteria, not on the leukocytes, and change them in such a way that they are more readily taken up by the phagocytes. Wright proposed for these bodies the name opsonin, derived from a Greek word signifying “to prepare a meal for.” Neufeld and Rimpau at about the same time (1904), in studying immune sera, observed substances of similar action in these sera and proposed the name bacteriotropins, or bacteriotropic substances. There is scarcely a doubt that the two names are applied to identical substances and that Wright’s name opsonin should have preference.
The chemical nature of opsonins is not certainly determined, but they appear to be a distinct class of antibodies and to possess two groups, a combining or haptophore and a preparing or opsonic group and hence are similar to antibodies of Ehrlich’s second order—agglutinins and precipitins. Wright also showed that opsonins are just as specific as agglutinins are—that is, a micrococcus opsonin prepares micrococci only for phagocytosis and not streptococci or any other bacteria.
Wright showed that opsonins for many bacteria are present in normal serum and that in the serum of an animal which has been immunized against such bacteria the opsonins are increased in amount. Also that in a person infected with certain bacteria the opsonins are either increased or diminished, depending on whether the progress of the infection is favorable or unfavorable. The opsonic power of a serum normal or otherwise is determined by mixing an emulsion of fresh leukocytes in normal saline solution with a suspension of the bacteria and with the serum to be tested. The leukocytes must first be washed in several changes of normal salt solution to free them from any adherent plasma or serum. The mixture is incubated for about fifteen minutes and then slides are made, stained with a good differential blood stain, Wright’s or other, and the average number of bacteria taken up by at least fifty phagocytes taken in order in a field is determined by counting under the microscope. The number so obtained Wright calls the phagocytic index of the serum tested. The phagocytic index of a given serum divided by the phagocytic index of a normal serum gives the opsonic index of the serum tested. Assuming the normal opsonic index to be 1, Wright asserts that in healthy individuals the range should be not more than from 0.8 to 1.2, and that an index below 0.8 may show a great susceptibility for the organism tested, infection with the given organism if derived from the individual, or improper dosage in case attempts have been made to immunize by using killed cultures, vaccines, of the organism.
On the occasion of the author’s visit to Wright’s clinic (1911) he stated that he used the determination of the opsonic index chiefly as a guide to the dosage in the use of vaccines.
Most workers outside the Wright school have failed to recognize any essential value of determinations of the opsonic index in the use of vaccines. Some of the reasons for this are as follows: The limit of error in phagocytic counts may be as great as 50 per cent. in different series of fifty, hence several hundred must be counted, which adds greatly to the tediousness and time involved; the variation in apparently healthy individuals is frequently great, hence the “normal” is too uncertain; finally the opsonic index and the clinical course of the disease do not by any means run parallel. Undoubtedly the method has decided value in the hands of an individual who makes opsonic determinations his chief work, as Wright’s assistants do, but it can scarcely be maintained at the present time that such determinations are necessary in vaccine therapy. Nevertheless that opsonins actually exist and that they play an essential part in phagocytosis, and hence in immunity, is now generally recognized.
Whether determinations of opsonic index are useful or not is largely a matter of individual opinion, but there is scarcely room to doubt that Wright has conferred a lasting benefit by his revival of the use of dead cultures of bacteria, bacterial vaccines, both for protective inoculation and for treatment. It is perhaps better to use the older terms “vaccination” and “vaccine” (though the cow, vacca, is not concerned) than to use Wright’s term “opsonic method” in this connection, bearing in mind that the idea of a vaccine is that it contains the causative organism of the infection as indicated on p. 253.
As early as 1880 Touissant proposed the use of dead cultures of bacteria to produce immunity. But because injections of such cultures were so frequently followed by abscess formation, doubtless due to the high temperatures used to kill the bacteria, the method was abandoned. Further, Pasteur and the French school persistently denied the possibility of success with such a procedure, and some of them even maintain this attitude at the present time. The successes of Wright and the English school which are being repeated so generally wherever properly attempted, leave no doubt in the unprejudiced of the very great value of the method and have unquestionably opened a most promising field both for preventive inoculation and for treatment in many infectious diseases. That the practice is no more universally applicable than are immune serums and that it has been and is still being grossly overexploited is undoubted.
The use of a vaccine is based on two fundamental principles. The first of these is that the cell introduced must not be in a condition to cause serious injury to the animal by its multiplication and consequent elaboration of injurious substances. The second is that, on the other hand, it must contain antigens in such condition that they will act as stimuli to the body cells to produce the necessary antibodies, whether these be opsonins, bactericidal substances, or anti-endotoxins. In the introduction of living organisms there is always more or less risk of the organism not being sufficiently attenuated and hence of the possibility of its producing too severe an infection. In using killed cultures, great care must be exercised in destroying the organisms, so that the antigens are not at the same time rendered inactive. Hence in the preparation of bacterial vaccines by Wright’s method the temperature and the length of time used to kill the bacteria are most important factors. This method is in general to grow the organisms on an agar medium, rub off the culture and emulsify in sterile normal salt solution (0.85 per cent. NaCl). The number of bacteria per cc. is determined by staining a slide made from a small volume of the emulsion mixed with an equal volume of human blood drawn from the finger and counting the relative number of bacteria and of red blood corpuscles. Since the corpuscles are normally 5,000,000 per c.mm., a simple calculation gives the number of bacteria. The emulsion of bacteria is then diluted so that a certain number of millions shall be contained in each cc., “standardized” as it is called, then heated to the proper temperature for the necessary time and it is ready for use. A preservative, as 0.5 per cent. phenol, tricresol, etc., is added unless the vaccine is to be used up at once. The amounts of culture, salt solution, etc., vary with the purpose for which the vaccine is to be used, from one or two agar slant cultures and a few cc. of solution, when a single animal is to be treated, to bulk agar cultures and liters of solution as in preparing antityphoid vaccine on a large scale.
Agar surface cultures are used so that there will be as little admixture of foreign protein as possible (see Anaphylaxis, p. 289 et seq.). Normal saline solution is isotonic with the body cells and hence is employed as the vehicle.
Lipovaccines.—The suspension of bacteria in neutral oil was first used by Le Moignac and Pinoy who gave the name “lipovaccines” (λιπος = fat) to them. It was claimed that the reaction following injection of these vaccines was less severe than with saline vaccines in many instances; also, that the bacteria were much more slowly absorbed. For these two reasons it was hoped that much larger numbers of bacteria could be injected at one dose and one injection would suffice instead of three or more as ordinarily used. The technique of preparation, standardization and killing of the organisms has not as yet been sufficiently well established to warrant the general substitution of lipovaccines for ordinary saline suspensions.
Vaccines are either “autogenous” or “stock.” An “autogenous” vaccine is a vaccine that is made from bacteria derived from the individual or animal which it is desired to vaccinate and contains not only the particular organism but the particular strain of that organism which is responsible for the lesion. Stock vaccines are made up from organisms like the infective agent in a given case but derived from some other person or animal or from laboratory cultures. Commercial vaccines are “stock” vaccines and are usually “polyvalent” or even “mixed.” A “polyvalent” vaccine contains several strains of the infective agent and a “mixed” contains several different organisms.
Stock vaccines have shown their value when used as preventive inoculations, notably so in typhoid fever in man, anthrax and black-leg in cattle. The author is strongly of the opinion, not only from the extended literature on the subject, but also from his own experience in animal, and especially in human cases, that stock vaccines are much inferior and much more uncertain in their action when used in the treatment of an infection, than are autogenous vaccines. This applies particularly to those instances in which pneumococci, streptococci, micrococci, and colon bacilli are the causative agents but to others as well. The following are some of the reasons for this opinion: The above organisms are notoriously extremely variable in their virulence. While there is no necessarily close connection between virulence and antigenic property, yet since virulence is so variable, it is rational to assume that antigenic property is also extremely variable. Individuals vary just as much in susceptibility and hence in reactive power, and generally speaking, an individual will react better in the production of antibodies to a stimulus to which he has been more or less subjected, i.e., to organisms derived from his own body.
In the preparation of a vaccine great care must be used in heating so that the organisms are killed, but the antigens are not destroyed. Many of the enzymes present in bacteria, especially the proteolytic ones, are not any more sensitive to heat than are the antigens, hence are not destroyed entirely. Therefore a vaccine kept in stock for a long time gradually has some of its antigens destroyed by the uninjured enzymes present with them, and so loses in potency. Therefore in treating a given infection it is well to make up a vaccine from the lesion, use three or four doses and if more are necessary make up a new vaccine.
If the above statements are borne in mind and vaccines are made and administered accordingly, the author is well satisfied that much better results will be secured.
In accordance with the theory on which the use of vaccines is based, i.e., that they stimulate the body cells to produce immunizing antibodies, it is clear that they are especially suitable in those infections in which the process is localized and should not be of much value in general infections. In the latter case the cells of the body are stimulated to produce antibodies by the circulating organisms, probably nearly to their limit, hence the introduction of more of the same organisms, capable of stimulating though dead, is apt to overtax the cells and do more harm than good. It is not possible to tell accurately when this limit is reached, but the clinical symptoms are a guide. If vaccines are used at all in general infections they should be given in the early stages and in small doses at first with close watch as to the effect. In localized infections only the cells in the immediate neighborhood are much stimulated, hence the introduction of a vaccine calls to their aid cells in the body generally, and much more of the resulting antibodies are carried to the lesion in question. Manifestly surgical procedures such as incision, drainage, washing away of dead and necrotic tissue with normal saline solution, not necessarily antiseptics, will aid the antibodies in their action and are to be recommended where indicated.
In the practical application of any remedy the dosage is most important. Unfortunately there is no accurate method of determining this with a vaccine. Wright recommended determining the number of the organisms per cc. as before mentioned, and his method or some modification of it is still in general use. From what was said with regard to variation, both in organisms and in individuals, it can be seen that the number of organisms is at least only a very rough guide. This is further illustrated by the doses of micrococcus (staphylococcus) vaccines recommended by different writers, which vary from 50,000,000 to 2,000,000,000 per cc. The author is decidedly of the opinion that there is no way of determining the dosage of a vaccine in the treatment of any given case except by the result of the first dose. Hence it is his practice to make vaccines of a particular organism of the same approximate strength, and to give a dose of a measured portion of a cubic centimeter, judging the amount by what the individual or animal can apparently withstand, without too violent a reaction. If there is no local or general reaction or if it is very slight and there is no effect on the lesion, the dose is too small. If there is a violent local reaction with severe constitutional symptoms clinically, and the lesion appears worse, the dose is too large. There should be some local reaction and some general, but not enough to cause more than a slight disturbance, easy to judge in human subjects, more difficult in animals. In cases suitable for vaccine treatment no serious results should follow from a properly prepared vaccine, though the process of healing may be delayed temporarily. Wright claimed, and many have substantiated him, that always following a vaccination there is a period when the resistance of the animal is diminished. This is called the “negative phase,” and Wright considered this to last as long as the opsonic index remained low, and when this latter began to increase the stage of the “positive” or favorable phase was reached. As has been stated the opsonic index is pretty generally regarded as of doubtful value, though the existence of a period of lowered resistance is theoretically probable from the fact that antibodies already present in the blood will be partially used up in uniting with the vaccine introduced and that the body cells are called upon suddenly to do an extra amount of work and it takes them some time to adapt themselves. This time, the “negative phase,” is much better determined by the clinical symptoms, general and especially local. It is good practice to begin with a dose relatively small. The result of this is an indication of the proper dosage and also prepares the patient for a larger one. The second dose should follow the first not sooner than three or four days, and should be five to seven days if the first reaction is severe. These directions are not very definite, but clinical experience to date justifies them. It is worth the time and money to one who wishes to use vaccines to learn from one who has had experience both in making and administering them, and then to remember that each patient is an individual case, for the use of vaccines as well as for any other kind of treatment.
Opsonins have been shown to be specific substances which act on bacteria in such a way as to render them more readily taken up by the leukocytes. By analogy one might expect to find bacteria secreting specific substances which would tend to counteract the destructive action of the phagocytes and bactericidal substances. Bail and his co-workers claim to have demonstrated such substances in exudates in certain diseases and have given the distinctive name “aggressins” to them. By injecting an animal with “aggressins,” antiaggressins are produced which counteract their effects and thus enable the bacteria to be destroyed. The existence of such specific bodies is not generally accepted as proved. The prevailing idea is that bacteria protect themselves in any given case by the various toxic substances that they produce, and that “aggressins” as a special class of substances are not formed.