Fundamentals and Possibilities in Anti-Tuberculosis Vaccination 9781487573805

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Fundamentals and Possibilities in Anti-Tuberculosis Vaccination

FUNDAMENTALS AND POSSIBILITIES IN ANTI-TUBERCULOSIS VACCINATION by Professor

RICHARD PRIGGE M.D., D.h.c. Director of the State Institute for Experimental Therapy, Paul Ehrlich Institute; of the Institute for Chemo-therapeutic Research, Georg Speyer House; and of the Ferdinand Blum Institute for Experimental Biology, at Frankfurt am Main and Professor

GUNTHER HEYMANN M.D. Scientific Member of the State Institute for Experimental Therapy, Paul Ehrlich Institute, at Frankfurt am Main Translated by

H. CHANDLER ELLIOTT Ph.D. Associate Professor of Anatomy University of Nebraska College of Medicine

UNIVERSITY OF TORONTO PRESS

University of Toronto Press Printed in Canada

Reprinted in 2018 ISBN 978-1-4875-7368-3 (paper)

[All rights reserved, 1960)

This translation of Grundlagen und Moglichkeiten der Tuberkuloseschutzimpfung is published with the permission of the original publishers Urban & Schwarzenberg, Munich • Berlin - Vienna

Preface Since the end of World War II, in discussion of the problem of protective vaccination against tuberculosis, BCG has almost exclusively held the centre of the stage. As a result it is often overlooked that, in tuberculosis, inoculation with attenuated living agents is not the only method by means of which immunity can be induced. The fact is also generally disregarded that opinions by no means agree as to the value, or lack of it, of BCG vaccination; particularly in the field of veterinary medicine, an almost entirely unfavourable evaluation of the procedure has recently prevailed. The reasons for dissent by veterinary doctors may, of course, be strongly influenced by the fact that they have available simpler and more radical methods of eliminating infection in livestock; yet a close study of the literature shows that the concepts of doctors in the human field not only disagree but also change. Here, outstandingly, we should recall the altered position of Wallgren, who formerly expressed himself unreservedly on behalf of general application of BCG vaccination. Corresponding to these facts, BCG vaccination today is no longer accepted to such an emphatic degree as a method suited to mandatory application, as was the case a few years back. Hence, investigations with the objective of improving or replacing the BCG method, are today once more claiming greater attention, especially in Germany. About eleven years ago, one of us, at the request of the editorial board of the Medizinische Klinik, presented a review on current problems in the field of vaccination in tuberculosis. It now seems timely to expand this summary, and to review and correlate the extensive experimental and clinical material that has been published in the interval. With this in mind, we contemplate the publication of the present short monograph at this particular time when a new development seems to be making headway. We shall attempt to

Vl

PREFACE

collect the information thus far available, in concise form, and to provide a basis for further work. We should like in this place to express our gratitude to Professor Bockemilller, who has carefully advised us on the drawing-up of the chapter on immunological chemistry. We owe special thanks to Dr. C. 0. Siebenmann, Research Member, Connaught Medical Research Laboratories, University of Toronto, and to Mr. M. Jeanneret, Director of the University of Toronto Press, who have made possible the translation of our monograph and its publication in the English-speaking world.

Frankfurt a.M., April 1959

R. PRIGGE G.HEYMANN

Contents PREFACE

V

1.

RESISTANCE

3

2.

IMMUNITY

6

3.

ALLERGY

15

4.

IMMUNOCHEMISTRY OF THE TUBERCLE BACILLUS

23 23 28 29 30 32 37

1. Proteins

2. Lipoids

(a) Acetone-soluble fats (b) Phosphatides (c) Waxes 3. Polysaccharides

5.

THE RELATIONSHIPS BETWEEN ALLERGY AND IMMUNITY

41

6.

METHODS OF VACCINATION

49 49 50 50 52 75

1. Vaccination with Fully Virulent Tubercle Bacilli

2. Vaccination with Attenuated Tubercle Bacilli

(a) Early experiments (b) BCG (c) Vole bacillus 3. Vaccination with Killed Tubercle Bacilli 4. Vaccination with Body Components or Metabolic Products of Tubercle Bacilli

7.

77

82

SUMMARY AND OUTLOOK

86

BIBLIOGRAPHY

89

SUBJECT INDEX

107

Fundamentals and Possibilities in Anti-Tuberculosis Vaccination

1. Resistance Man and those mammals most susceptible to tuberculosis (e.g., monkeys and guinea-pigs) possess a more or less marked power of resistance against the tubercle bacillus. This is a characteristic present even before contact with the pathogenic agent, and it varies greatly, individually and in time; opinions diverge widely on its extent. On behalf of the thesis that the genus Homo is generally susceptible to the tubercle bacillus, Doerr2 has adduced the fact that more than 95 per cent of the total population become tuberculin-positive in the course of their lives; but arguments are presented for the view that even if a general susceptibility exists, nevertheless a high percentage (3o-40 per cent according to Geissler) is resistant to tuberculosis infection; this opinion is also manifestly concurred in by clinicians (lckert3). The accidental experiment in Lubeck-the feeding of more than 250 infants with doses of virulent human tubercle bacilli, some of them massive-resulted, according to Moegling and Zoelch, in fatal tuberculosis in only 27 per cent of cases, in disease with remission in 56 per cent, and in mere tuberculin allergy without disease in 17 per cent. One should take into account that we are here dealing with a group of individuals whose primary powers of resistance are especially slight. Thus, one can draw from the results of even this regrettable mishap, a certain confirmation of Geissler's position. As a determining factor in tuberculosis, resistance enjoys a material significance. This has been given recognition in its full implications, particularly through the works of Kleinschmidt and Bruno Lange2 • The genetic determination of resistance was established by Diehl and Verschuer by a comprehensive collection of tuberculosis cases in twins. In 80 identical pairs, 65 per cent showed similar, 35 per cent dissimilar tuberculous conditions; in 125 fraternal pairs, only 25 per cent were found with similar, 75 per cent with dissimilar conditions.

4

ANTI-TUBERCULOSIS VACCINATION

The investigations of Dehlinger and Kunsch, of Kallmann and Reisner, and of Vaccarezza and Dutrey, add up to a material of altogether more than 600 pairs of twins, showing concordant tuberculous conditions in 74 per cent of cases in identical twins, 28 per cent in fraternal twins (Verschuer2 •3). A follow-up investigation, after more than twenty years, has been undertaken by Mitschrich on 118 of the 239 pairs of twins previously recorded by Diehl and Verschuer. This shows that the further course of the disease also confirms the conclusions drawn by the authors. The concordance of the identical pairs, and the discordance of the fraternal pairs, have actually become even more marked. The investigations of Diehl through experiments with animals, on the hereditary determination of resistance to tuberculosis, led to conclusions similar to those of the observations on twins. A "lungresistant" (Verschuer 1) strain of rabbits was found which, after tuberculous infection, perished from peripherally localized lesions, and a "lung-susceptible (central)" strain. Cross-breeding experiments produced only the lung-resistant type in the F 1 generation, which divided in the F2 generation in a 3:1 proportion. From this, Diehl deduced a simple mendelian inheritance, in which the "lung-resistant" type is dominant over the central type. The decisive significance of resistance in the inception and course of tuberculous infection is often not recognized even today. Yet obviously it bears on the possibility of effecting protective immunization against tuberculosis and has especially to be taken into account in the statistical evaluation of the results of vaccination. On this point, the investigations of Lurie4 •5 and of Lurie, Zappasodi and Tickner, give clear evidence. They not only confirm the hereditary determination of resistance, but also point out the possibility that endogenous factors interfere with immunizing processes. Thus, for example, unvaccinated rabbits of a resistant strain showed a far higher ability to withstand bovine infection, than did animals from a population with genetically lower natural resistance though protectively vaccinated with human tubercle bacilli (Lurie cited by Diehl). Furthermore, at the same time it became evident that the reaction of rabbits to intracutaneous injections of BCG could actually serve as an indicator of natural resistance (Lurie, Zappasodi, Cardona-Lynch and Dannenberg). In cases of high resistance, the reaction at the point of vaccination proceeds rapidly; it shows a tendency to ulceration and heals with connective-tissue scar and elimination of the tubercle bacilli, with a quickly mounting allergy to tuberculin. Mononuclears

RESISTANCE

5

predominate in the blood. In animals with low natural resistance, the course of the local reaction is prolonged, without ulceration, and passes over into caseation rich in tubercle bacilli. In the blood, polymorphonuclear leucocytes are prevalent. Whether this difference in reaction can also serve to demonstrate the degree of resistance in man, remains to be seen. We may hold as undecided to what extent, compared with resistance, other endogenous conditioning factors-such as hormones (Schafer, Tonutti), age (Klare), functionally (Derbalow) and nervously determined influences-play a part in the course of tuberculosis. In sum, we can probably say that environmental influences, however powerfully they may bear on the individual case, do not by far play that decisive role in the course of tuberculosis that has been, for so long, attributed to them.

2. Immunity Immunity, arising only as a reaction to tuberculous infection, must be clearly distinguished in concept from natural resistance. Our essential knowledge on the phenomena of tubercular immunity we owe to Robert Koch; and any discussion on the subject must start from the experiment that has become famous as Koch's basic experiment or Koch's phenomenon. This was described by Koch 1 in the following words: If we inoculate a healthy guinea-pig with a pure culture of tubercle bacilli, the inoculation wound normally closes and appears to heal in the first few days; only in the course of 10-14 days does a hard nodule arise, which soon ruptures and forms a point of ulceration up until the death of the animal. But matters proceed quite otherwise if we inoculate a guinea-pig already sick with tuberculosis. Animals which have been successfully inoculated 4-6 weeks previously are particularly suitable for this purpose. With such animals, the small inoculation wound also closes initially, but no nodule forms; rather, on the very next day or the second day, a characteristic change sets in at the point of inoculation. This grows hard and takes on a darker colouration; which, indeed, does not confine itself to the point of inoculation alone, but expands into the vicinity up to a diameter of 0.5-1 cm. On the following days, it appears ever more clearly that the skin thus altered is necrotic; it is finally sloughed off, leaving behind a superficial ulceration, which generally heals rapidly and permanently, without infiltration of the neighbouring lymph nodes. The inoculated tubercle bacilli thus act quite differently on the skin of a healthy and that of a tuberculous guinea-pig.

Of course, the tubercle bacilli introduced in the second infection are not destroyed in the immune organism, but are only restrained from multiplying. However, they retain their virulence: if they are reinoculated into normal animals, the latter regularly sicken with tuberculosis (Willis). Furthermore, the protection bestowed by the immunity acquired during an attack of tuberculosis is only limited. A second (super)

IMMUNITY

7

infection, undertaken with a small number of bacteria, may give rise to only trivial tuberculous changes or proceed completely without symptoms (when dealing with an "infectio minima" which does not exceed the degree of the primary infection; B. Lange4); but large numbers of bacteria break through the protection and lead, always in proportion to the dosage, to more or less severe reactions (Romer 3 , Hamburger). This may also explain why the immunizing processes often do not suffice to suppress regularly and adequately the multiplication of the germs originating from the primary infection: the number of the germs overwhelming the organism in a bacterial invasion is too great in proportion to the powers of defence. The contradiction which apparently arises here cannot, however, be explained by the characteristics of the tubercle bacillus. In particular, the assumption that the tubercle bacillus undergoes qualitative changes in the infected organism, which might bestow heightened powers of resistance upon it, has been refuted by the evidence provided by Romer and Joseph; they showed that tuberculous animals are protected in the same way against tubercle bacilli taken from their own bodies and re-injected at different points, as against other germs. Also, the temporal duration of protection against fresh infection is limited. This protection is regarded by almost all investigators as infection-(or relative) immunity; i.e., it persists only as long as the agents of the primary infection reside in the body, and vanishes as soon as these are eliminated or the disease is "cured." Even though true cures are not so rare as is generally supposed (Keller), the majority of people who were once infected with tuberculosis, but who did not suffer progression of the disease, still harbour residual tubercle bacilli all their lives. The latter live in a true symbiosis with the host organism, and can lead to an exacerbated infection only if a lowering of the power of resistance occurs through exogenous or endogenous causes (Bloch 2 ) or through other disturbances of the biological equilibrium. This continuing stimulation is undoubtedly the source of the immunity against exogenous superinfection found in most people. A concept has been put forward in recent years in Russian circles (Kagramanov), according to which a persistence of tubercle bacilli or the presence of tuberculous tissue is not necessary for the establishment of a specific immunity. This is in opposition to the views of "Western" schools, even though Bloch2 concedes that their concepts as to the establishment of relative immunity, are no more than a working hypothesis at present.

8

ANTI-TUBERCULOSIS VACCINATION

Kagramanov's principles are based on Pawlow's doctrine, transferred to tuberculosis. This postulates a stimulation of nervous receptors in the lymphatic and reticulo-endothelial systems, which sets in at the time of infection and is accompanied by a chain of biochemical reactions, and then maintains immunity to tuberculosis even if tubercle bacilli and specific tissue are eliminated from the organism. This continuing readiness for reaction of the nervous system manifests itself as a defence against disease, in the form of cellular phagocytic reactions as well as (poorly understood) humoral mechanisms. Undoubtedly, the autonomic nervous system can play a not insignificant role in the genesis and maintenance of immunity (cf. Enenkel), even in tuberculosis. On this point, Catel and Daelen, and lckert4 have given emphatic testimony, based on the work of Speransky, Sturm, Nonnenbruch and Stohr Jr. (cited by Cate! and Daelen 1); so also have Schwartz and Waitz, and a number of Russian authors (cf. Eitner) . Nevertheless, we should no more over-emphasize the significance of nervous influences on immunity, than others should have overemphasized, in its time, the immuno-biological viewpoint on the course of tuberculosis going back in particular to the work of Ranke. There are arguments enough to suggest that we should evaluate cautiously the role of the vegetative nervous system in the dynamics of organic response to stimuli (Engelhardt and Lendel), even if we do not altogether adopt the judgement of Ehrich which recently designated Ricker's step-law as "untenable." The maintenance and degree of the immunity certainly depend largely on the permanence and degree of the stimulus emanating from the tubercle bacilli. This reaction status, unusual in the biology of immunity, leads to the phenomenon, formerly observed by B. Lange,' of "association of high immunity with severe and progressive, and low with regressive tuberculosis" (B. Lange, see p. 312). It is also one of the reasons why tuberculous immunity is regarded by a number of authors as a special case of general immunity, or is altogether denied, as it is by Schmid 5, who recognizes neither a true nor a relative immunity in tuberculosis. From the established fact "that neither symptomless tuberculous infections (with positive tuberculin reaction) nor clinically healed cases of tuberculosis show evidence of an after-effect of immunization, i.e., protection against re-infection," Blittersdorf goes so far as to draw

IMMUNITY

9

the rather unconvincing conclusion that "vaccination immunity" is not a specific phenomenon but is based on the effect of an "interference" (Freerksen) restricting superinfections. Here, Blittersdorf overlooks the fact that, in almost all infectious diseases, sooner or later exogenous re-infections occur and can lead to renewed sickness, after the antigenic stimulus emanating from the pathogens of the primary infection has ceased or sharply declined as a result of the pathogens' disappearance or transition into latency. Bloch's2 recent report, introduced with the question "Is there an immunity against tuberculosis?" also expresses the general uncertainty in this field. Neither the comprehensive experiments with BCGvaccination nor clinical or epidemiological investigations have been able to provide a solution for the question thus posed (Bloch 2), even though the answer to this very question is crucial for the possibilities of anti-tuberculosis vaccination. Concepts of the origin, degree, and mechanism of specific prevention of tuberculosis are in part highly speculative. Experimental material has been presented for the most diverse theories. The way in which tuberculous immunity appears certainly strongly resembles in many points the phenomena appearing after other forms of antibacterial, active immunization; this is particularly true as regards its course in time or dependency on dosage (Bloch 2). Nevertheless, substantial divergences appear in its fundamental immunological characteristics, compared with ordinary antibacterial immunity. The latter, almost without exception, makes itself evident by the appearance of free antibodies, the activity of which is demonstrable even in vitro. By contrast, with immunity against tuberculosis the role played by the antibodies which develop is still quite unclear and is rated very low by many authors (Cate!) if they assume that these antibodies have any specific function at all. The fact that tuberculosis immunity cannot be transferred to normal experimental animals by the serum of infected or otherwise immunized individuals, plays the decisive role in these considerations. On the other hand, the demonstrability of humoral antibodies in the serum of tuberculous subjects admonishes us to caution; this being deducible from complement fixation, agglutination, precipitation (cf. Pfannenstiel), or indirect haemagglutination (Middlebrook and Dubos; Gernez-Rieux and Tacquet), and from the possibility, provided by the latter, of drawing conclusions as to the immunological status of the subject or the degree of activity of his disease. New possibilities now appear to arise in this connection, particularly from

10

ANTI-TUBERCULOSIS VACCINATION

the recently presented method of Gerstl, Davies, Kirsh, Hollander, Barbieri and Weinstein. This method, which combines Cohn's plasma fractionation with the technique of indirect haemagglutination, is claimed to be capable of demonstrating the presence of antibodies in cases of active tuberculosis, when other methods fail to do so. The work of Lurie13 based on animal experiments, also notably supports the concept of the contribution of humoral antibodies to tubercular immunity. Two of his experimental series gave particularly impressive results: an infusion of tubercle bacilli in agar, injected into the experimental animal subcutaneously and jellying rapidly in the body, becomes saturated with the body fluids whereas cellular elements can penetrate it only very slowly. In previously untreated animals, the growth of the tubercle bacteria was promoted, in tuberculous animals, it was restricted. Similarly, when suspensions of tubercle bacilli were introduced into the peritoneal cavity of rabbits by means of collodion sacs, impermeable to cells, the growth of the bacilli was promoted in normal animals, restricted in tuberculous ones. At the same time, a distinct clumping of the bacteria was observed in the tuberculous animals. Of great interest in this connection, is the recent demonstration by Seibert, Miller, Busemann, Seibert, Soto-Figueroa, and Fry, and by Seibert and Seibert, of a relationship of anti-tuberculous immunity with antibodies in the serum of tuberculous animals; the antibodies were directed against tuberculoproteins and polysaccharides and demonstrable by precipitation and complement fixation. Hence, evidently the presence of a well-balanced antibody spectrum is the prerequisite for the establishment of a specific immunity. In rabbits with known, genetically conditioned lack of resistance, anti-polysaccharides were lacking after immunization, whereas rabbits with great natural resistance developed through BCG immunization a complete antibody spectrum with high titre values. Lurie, Zappasodi, and Tickner had already made similar observations. Furthermore, the investigations of Seibert and co-workers (see above), showed that tuberculo-polysaccharides themselves are in a position to act restrictively in the immune system, only when they are present in excess and cannot be taken up by homologous antibodies. The latter must be the case in the rabbits that are incapable of forming anti-polysaccharides. This assumption can be proved by the marked titre-reduction of the full antibody-spectrum upon administration of polysaccharides.

IMMUNITY

11

Tuberculo-polysaccharides have an ability to disturb specific antigen-antibody relationships when homologous antibodies are lacking, as does Seibert's Protein A in a lesser degree (see chap. IV, 1, p. 27). This could be an explanation for the fact that, in spite of immunization and the formation of humoral antibodies, frequently no power of resistance against tuberculosis develops. In analogy to the attempts to passively transfer tuberculin allergy (cf. p. 19), Zitrin and Wasz-Hockert carried out investigations on the possibility of passively transferring protective antibodies with serum fractions or whole serum of tuberculous subjects or with immune gamma-globulin; these are supposed to have effected, in vitro, a growth restriction of H 37 Rv and, in vivo, a prolongation of the time of survival in mice infected with the Valle strain. These and a number of further experimental results (Ghara and Nakagawa; Ohara and Nakagawa) and clinical reports testify to a participation of humoral antibodies in tuberculous immunity. And probably no further discussion is required to establish that they do take part along with natural resistance and cellular defence processes, in the defence against the invading and established pathogens, though the transfer of these humoral antibodies to normal animals is by no means sufficient to bestow upon these a protection against the invasion and multiplication of tubercle bacilli. The extent and significance of cellular defence is likewise very diversely estimated (Mackaness). The responsibility of throwing off primary infection appears to fall in the first place on the reticuloendothelial system; and this can develop restrictive powers associated with specific tissue transformation as immunity builds up. According to Lurie, it is the mononuclear phagocytes that play an important role besides other protective mechanisms already mentioned. Many authors (Lucke, Strumia, Mudd, McCutcheon and Mudd, Woodruff, Thomas, Smithburn) recognize the alterations in the reactive properties of these cells arising under the influence of the first infection and leading to an ability to quickly phagocytose and resorb tubercle bacilli. In particular, the observations of Lurie confirm earlier works of Smithburn and Sabin 1• According to Lurie, the mononuclear phagocytes take on, with mounting immunity of the organism, the behaviour and structure of epithelioid cells; Smithburn and Sabin established that, with resorption of increasingly more finely dispersed phosphatides from tubercle bacilli, monocytes attain the character of epithelioid cells.

12

ANTI-TUBERCULOSIS VACCINATION

The significance of the leucocytes for the status of immunity or the hyperergic reaction has been repeatedly demonstrated both clinically (Langner) and also, more recently, by isotope investigations (Ritts and Favour) . Here the question necessarily arises, whether cellular defence is a matter of autonomically directed processes independent of immune bodies, or whether the changed mode of reaction of the mononuclears is the result of the formation of sessile antibodies. In other words: in just this field of defence against infection, the points of view of relational pathology and of classical immuno-biology meet; and various authors have for long assumed a close co-operation between sessile antibodies working through cellular defence and non-specific mechanisms (Kall6s; Spink and Keefer ; Zinsser, Enders, and Fothergill) . A final clarification of this question is not to be expected in the foreseeable future. Up until today, it has not been possible, even when specific immune bodies are clearly present, to demonstrate by chemical means anything like an equivalent for the antibody function of globulin, though it undoubtedly must be present-even if it is connected merely with a change in chemical configuration. To determine the material basis for the change in mode of reaction in the cell would be far more difficult yet. The observations of Heimbeck1. 3 and Dahl, made on a large number of individuals, were especially illuminating with regard to the state of immunity in man. They established that nurses who had a negative tuberculin reaction at the beginning of their student life fell sick and died from tuberculosis during work in an infectious environment, with disproportionately greater frequency than those with positive reactions or already infected with tuberculosis at the inception of their activity. In rebuttal of the conclusions to be drawn from this, one can, of course, raise an objection. Among the infected cases, only those could follow their vocation who had already come to terms with the infectious agents and, hence, individuals in danger from the disease simply did not appear any more among the student nurses in the tuberculin-positive group. Consequently, it would not be surprising if a group having passed through a "selection" of this sort should prove to be resistant towards further infections also. Contrary to the view of Grumbach and agreeing with that of Freudenberg, one can attribute no greater significance to the morbidity than to the mortality figures in dealing with this objection. Nevertheless, the findings of Heimbeck and Dahl have a fundamental significance. For proper evaluation of the circumstances, one must proceed from this consideration: that the proportion of

IMMUNITY

13

tuberculin-negative nurses who fall sick when exposed to more frequent, more massive infections is very much higher than the proportion of persons who fall sick from the weak infections that are the rule in normal circumstances. This being so, we might have expected that the tuberculin-positive, that is infected, student nurses who had nevertheless remained healthy under the conditions of their previous lives, would have sickened in a higher proportion under the conditions of a tuberculosis sanatorium-indeed, not much less than the tuberculinnegative nurses; unless, precisely, the slight infection they had undergone before entering their training had led to a change in their susceptibility to infection. Conclusions tending in the same direction as those of Heimbeck and Dahl have been drawn by numerous other authors, particularly by Madsen, Holm and Jensen; by Ferguson 1 ; by Urech and Rochat; by Mikol, Horton, Lincoln and Stokes; by Scheel; and also by Lunn who observed an unfavourable outcome in 95 per cent of the cases, in 37 acutely tuberculous children with negative tuberculin reactionswhich, even for the most susceptible age-groups, represents a lethality far above the average for tuberculin-positive patients. On the other hand, however, we have reports of an opposite immune status particularly by Chausinand, by Karasu (cited by Keller and Schomerus), and by Silviera3 • According to these, of the total investigated series of exposed persons those who sickened had become tuberculin-positive either spontaneously or after BCG-immunization; whereas those persons who were still tuberculin-negative (Chausinand), or had become negative again (Karasu), or had been made negative by BCG-desensitization, did not become tuberculous in spite of exposure. The significance of these findings, from which-especially in South America-even practical conclusions have been drawn concerning BCG-immunization, will be discussed in chapter V. Up to now, we still lack thoroughly satisfactory investigations which have been carried out with unimpeachable statistical methods, and which sufficiently take into account, in assembling the series to be compared, the numerical dislocations due to the above-mentioned "selections." The relationships between natural resistance and acquired immunity have also aroused special interest, as is evident from the work of Lewis and Loomis. On the basis of their investigations on infected guinea-pig populations, Wright and Lewis believed they could correlate the degree of resistance of various families with their capabilities for antibody

14

ANTI-TUBERCULOSIS VACCINATION

formation. This assumption was supported by the works of Seibert, Miller, Seibert, Soto-Figueroa and Fry; Tabeyi, Shiomi and Fukazawa claim likewise that the degree of capability to respond to an antigenic stimulus (infection, vaccination) with the development of an immunity is dependent upon natural resistance. Lurie2 , Lurie, Abramson and Heppleston reached conclusions similar to those of Wright and Lewis, using various strains of rabbits exposed to airborne infection. In the resistant animals, the individual cells possess the ability to eliminate the tubercle bacilli that have invaded the organism, apparently as a result of the ability to produce sessile antibodies rapidly. These conclusions suggest an association of the conditions "resistance" and "immunity," which are conceptually so clearly distinguished. In any case, the two principles of defence against infection cannot always be demarcated from each other in the individual example (Wallgren7). Whether their mechanisms are identical, as assumed by Bernard, by Cummins, and by Rich 3 , or at least closely linked (Kawamura), is nevertheless questionable, and a number of weighty arguments speak against it (Wallgren7 ).

3. Allergy Koch's fundamental discovery led not only to the revelation that the tuberculous organism acquires a defence against a second infection; at the same time, it demonstrated a close relationship of this defence with the development of a hypersensitivity against body fragments or metabolic products of the tubercle bacillus-what today, with Von Pirquet, we mostly call an allergy or, more precisely, a hyperergy. Even with dead tubercle bacilli, a conditioning to allergic reaction can be induced (Bessau 2 ; Romer2 ; Uhlenhuth and Jotten), whereas an immunity supposedly does not develop (Monaldi 1 ; Palmer and Edwards); this concept-so far as the comparison with living BCG goes-could not be substantiated by experiments with animals (Helmert; Langer; PetrofP; Schroder and Crawford). It is little known that animals with tuberculous allergy succumb immediately or within 24 hours after intravenous or intraperitoneal injection of great numbers of tubercle bacilli (10-100 mg.) or of tuberculin (Hart, Long, and Rees; Jeney; Kirchheimer and Malkiel; Maschmann and Kuster; Urbach), while in normal animals no severe reactions can be observed at first. The occasional delay of the "tuberculous shock" occurring after reinjection of bacteria, compared with the usual anaphylactic shock, is probably to be referred to the corpuscular character of the antigen (Kall6s and Kall6s-Deffner, cited from lckert2). After an episode of this sort, then, not only is protection against a new infection "breached," but phenomena of hypersensitivity dominate the picture completely. In searching for the substances which produce the reactions of hypersensitivity, Koch succeeded in demonstrating that they are contained in especially high concentration in tuberculin, i.e., in filtrates from cultures of bacteria, rendered germ-free and concentrated for several hours at temperatures up to 90°C. Koch himself divided the effects produced by tuberculin in the hypersensitive tuberculous

16

ANTl-TUBERCULOSIS VACCINATlON

organism into local, focal, and general reactions. For the uninfected body, tuberculin is notoriously indifferent; upon this rests the unique value of tuberculin for the detection of the tuberculous infection. Hence, tuberculin is not a "poison," and so ought not to be counted among the toxic compounds, the "toxins" or "endotoxins" formed by other infectious agents, e.g., the diphtheria or dysentery organisms. On the contrary, the toxic lipoids isolated by Bloch, Sorkin, and Erlenmeyer, by Noll and Bloch, by Asselineau, Bloch and Lederer, and by Spitznagel and Dubos, from the wax-fraction of the tubercle bacilli -Bloch's "cord-factor" and Spitznagel's "fraction 7"-are at least chemically similar to the endotoxins of the gram-negative bacteria (lipo-polysaccharides). Of course, the toxic compounds isolated from tubercle bacilli are not to be compared immunologically with the endotoxins. On grounds of pathological-anatomical findings also, one must decline to identify them with endotoxins (Humbert). To understand the nature of the substances responsible for the specific tuberculous allergy, we must here insert a few general comments. Acquired immunity generally finds expression in the fact that substances appear in the serum which are able to react in characteristic fashion with the disease agents or . their toxins and to render them harmless; the former are designated as protective substances or antibodies. We conversely designate as antibody-formers or antigens, substances the parenteral administration of which incites the formation of antibodies. It has now been shown that not only bacteria and their toxins or similar noxious substances, but also many neutral substances, are antigens; in particular nearly all proteins, as long as they are not present in the system of the pretreated individual; hence they induce the development of antibodies after subcutaneous, intraperitoneal, intravenous, or other methods of parenteral injection. On the other hand, the unique state of affairs arises that in men or animals into whom the active protein has been injected, after about 10-14 days a strong hypersensitivity arises to the antigen administered in the pretreatment, though this is in itself entirely non-toxic. Whereas, for example, a normal guinea-pig tolerates the injection of massive doses of bovine serum, a sensitized animal (i.e., pretreated with bovine serum) reacts with severe, often fatally terminating, convulsive fits ("anaphylaxis") on a repeated parenteral injection of the same material, and especially on intravenous administration. Also in the skin of animals that have become hypersensitive, the antigen used in the pretreatment induces, at the point of reinjection, violent in-

ALLERGY

17

flammatory phenomena which generally lead to necrosis and sloughing of the area of skin involved ("Arthus phenomenon"). It is accepted as proven today, that the phenomena of hypersensitivity described depend on the reaction between antigen and antibody, and even predominantly on that part of the process proceeding intracellularly; that is, they are conditioned by antibodies that are fixed in the cells (Bibi. in Doerr 2). In specific cases, the blood contains no antibodies at all or only such as can be demonstrated with special methods. As for true antigens it is considered that they not only react with the corresponding antibodies, but also have the power to induce formation of these antibodies in the living organism. But there are also compounds which are indeed capable of reacting with the antibody already present, but are not able to induce readily formation of this antibody; these we call "haptens." Among these belong, for example, many denatured albumins, certain carbohydrates of bacterial origin, pre-eminently the polysaccharides isolated from the capsular material of pneumococci, as well as numerous lipoids. These compounds act as full antigens only in conjunction with the natural cells; nevertheless, the antigenic activity of the haptens can in many instances be re-established by formation of compounds with albumins (Moro and Keller), erythrocytes (Weidmann), or by attachment to appropriate adsorbing materials (kaolin, collodion). These general basic rules are valid-with a few peculiarities-for tuberculous allergy also. This arises in the course of the tuberculous infection or after artificial immunization with attenuated or killed pathogens. And it is dependent temporally and in degree on the individual responsiveness of the immunized individual as well as on the extent of the infection and the virulence of the infecting bacteria. The appearance of the allergy terminates, in a sense, the incubation period and leads to the clinical symptoms of the primary complex, to the formation of specific tissue, and, in certain circumstances, caseous necroses. In practice, intracutaneous administration of tuberculin serves to establish the status of allergic reaction, assuming, no doubt correctly, that the skin allergy runs parallel to the general conversion (Tison). Hence, a localized Koch phenomenon is regarded as indicating the presence of a tuberculous infection, even though various authors have indicated the possibility of non-specific reactions (Palmer3 , Schliesser) and demonstrated the questionable nature of the method (Haase); they may base this on inadequacies of tuberculin and also on a deviation of the reaction-status of the organism (Canetti).

18

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The morphological specificity of the tuberculin reaction is diversely assessed. Schmid 3 was unable to demonstrate tuberculous tissue in the focus of inflammation; Miescher considers allergic reactions in general to be histologically non-characteristic; and also Hori could establish no sure histological distinction between the Arthus phenomenon and the tuberculin reaction, whereas Cate! and Wurm, and ReichelBergmann observed isolated epithelioid cells and giant cells of Langhans. These "foreign-body granulomas" can, of course, also be produced "non-specifically," for example by certain long-chain and branched fatty acids (Lederer). Induction of tuberculous-specific tissue does not appear necessarily to take place, and must certainly be evaluated differently for various animal species (Gray and Jennings). On the other hand, the concept maintained by Huth (after Cate! and Schmid) and shared by many authors, that the tuberculin reaction is primarily produced by histamine set free as a result of the antigenantibody reaction, also could not be confirmed by Cate! and Schmid, by Lima and Rocha, or by Neumann 2 • However-as in the establishment of immunity-the autonomic nervous system appears to play a certain though not fully understood role (Cate! and Schmid; Model). From the moment the tuberculous allergy develops, every superinfection is responded to by a Koch reaction analogous to the skin reaction; this can be taken as a blocking process against the spread of a new infection (Etzler). Phagocytosis, predominantly by mononuclears, sets in rapidly; the multiplication of the penetrating bacteria is restricted, and in their spread hindered (Wallgren7) ; thus the regional lymph nodes, in general, are mostly not even reached any more by the infection. The close relationship of allergy and rising power of resistance on the one hand with the occurrence of macrophages on the other has recently been pointed out again by Gray and Jennings. In investigations very recently conducted by Sternberg and Frappier, using isotope-labelled tubercle bacilli (32 P), animals pre-infected with BCG showed a distinctly lower incidence of bacteria in their organs than did control animals, after injection of an equal number of bacteria. The mobilization of the cellular defence mechanisms, which sets in almost abruptly leads to a sensitization of all cells; this has led almost forcibly to the conclusion-especially for investigators oriented towards relational pathology-that the establishment of allergy must be regulated by the autonomic nervous system (lckert4, Schwarz). The question of the correctness of this assumption may be left open.

ALLERGY

19

Nervous influences can be no more excluded from the hyperergic manifestations of allergy than from the immunological. Nevertheless, we may consider the fact as established, that the hyperergic-including tuberculin-hyperergic-processes have antigen-antibody reactions as a basis, among which antibodies of cellular derivation must be assigned a share on the part of the organism. Landsteiner, as early as 1942, transferred a hyperergy, which had been induced in guinea-pigs, which was not serologically demonstrable, and which could not be transferred by serum, from sensitized to normal animals by means of peritoneal exudate. Thereafter, Chase, in 1945, succeeded in transferring tuberculin allergy by exudate, and also by spleen and lymphoid-tissue cells of animals with tuberculin allergy. These investigations have since been repeated and reproduced under various experimental conditions by, among others, Cummings, Hoyt and Gottshall, by Cummings, Patnode and Hudgins, by Kirchheimer, by Lawrence1- 3, by Metaxas and Metaxas-Buehler1, by Stavitsky, Oliveira-Lima, Kourilsky and Decroix, and by Schmid 1 who regards monocytic cells as those primarily transferring allergy. The parallel with the histology of specific defence against infection, in which the mononuclears likewise play a decisive role, is here obvious. Even with spinal fluid cells (Schmid 2) and lymphocytes from the thoracic duct (Wesslen 1) of guinea-pigs with tuberculous infection, tuberculin allergy can consistently be transferred passively. The same has been observed in man after blood transfusion, especially after exchange transfusion (Schlange). Cell-free body fluids (serum, transudates, and content of skin blisters) of individuals hyperergic to tuberculin (Magrini and Angelucci) and likewise granulocytes (Kirchheimer) are, on the contrary, said to be inactive. Transferable tuberculin allergy is usually of only short duration, and is furthermore dependent in degree on the number of transferred cells and on the degree of sensitization of the donor animal. Furthermore, the often excessively high doses of tuberculin necessary to demonstrate the transferred allergies also raise doubts whether we are here dealing with a true passive allergy in the classical sense. Yet, histologically, no difference can be seen between the build-up and decline of the acute allergic reaction in tuberculous and passively allergized animals (Wesslen). Although the mechanism of the phenomenon has not been fully clarified the idea is prevalent that sessile antibodies are responsible for transfer of the allergy. Indeed, we must assume a transfer of cell-

20

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borne antibodies to the cells considered to be carriers (mononuclears) in the host animal, since the short duration of the transferable tuberculin allergy makes improbable an incisive change in biological function in the cells of the normal animal (Schmid 1). Furthermore, the investigations of Roberts and Dixon testify to the formation of a so-called "secondary immune reaction," which was observed in the transfer of cells from lymph-nodes of immunized rabbits into X-ray irradiated hosts. The transferred cells formed further antibodies in these animals which were themselves incapable of forming antibodies; the amount of these, according to the authors' calculations, attained in the first 8 days 2/ 3 of the wet weight of the transferred cells. The chronicle of passive transference of tuberculin allergy will reach a sensational conclusion, if the recently presented work of Cole and Favour should be confirmed . Since the (isolated) claim of Zinsser and Mueller that tuberculin allergy was transferable by means of a humoral antibody, findings to this effect have so far not been reported again. Attempts by Chase to transfer tuberculin hyperergy by means of dead donor cells, were unsuccessful, whereas Lawrence2 , and Lawrence and Pappenheimer, were able to transfer allergy of the delayed reaction type (cf. chapter IV (1) p. 27) both against tuberculin and also against streptococcal substances and diphtheria toxin, with cell lysates and cautiously prepared leucocyte extracts. Cole and Favour have just recently succeeded in isolating two chemically different antibodies from the plasma of immunized and hyperergic guinea-pigs, by means of the fractionation method "X" developed by Cohn et al. These were: (1) An antibody ("II") which is located in the gamma-globulin and directed against the polysaccharide fraction of the tuberculin; it can be demonstrated by the haemagglutination technique of Middlebrook and Dubos, and is capable of transferring hyperergy of the Arthus phenomenon type, directed against tuberculo-polysaccharide, from immune to normal guineapigs. (2) An antibody ("IV-10") which is located in a new plasma fraction pertaining to alpha-globulin, and is directed against the protein fraction of tuberculin; it can be demonstrated serologically by means of Boyden's technique, and can passively sensitize normal animals against tuberculoprotein (PPD = purified protein derivative), in which case a characteristic tuberculin allergy of the delayed reaction type occurs. Shortly after the report of Cole and Favour (whose findings could not be reproduced by Ehrenkranz and Wachsman) there appeared a

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detailed work by Metaxas and Metaxas-Buehler2 , in which the "specific cells" were made primarily responsible for the development of the tuberculin allergy. If a mechanism conditioned by antibodies is conceded, then it is only in the form of a complete integration of the antibodies into the cellular structure. The discrepancy of the concepts concerning the mechanism of tuberculin allergy cannot be explained away even by the investigations of the same research circle on the cytotoxic activity of tuberculin on cells of hyperergic animals. These investigations served primarily as pilot experiments, but nevertheless realized a number of interesting findings. They were preferentially carried out on cell cultures, which offer the advantage that one can use homogeneous cell populations while excluding neural or vascular influences (Favour2 ). With the exception of a few authors (Lasfargues, also Baldridge) a specific damage to tissue cultures from tuberculous animals by tuberculin was generally confirmed (Aronson 1 ; Dittmar; Heilmann; Moen; Rich and Lewis). Of course, opinions are not entirely uniform as regards differentiation of the especially susceptible cells. Nevertheless, the work of Waksman, of Waksman, Carroll and Gaulitz, of Brand, Sandoge and Biekeland, of Fremont-Smith and Favour, and of Schmid4, reveals that it is predominantly macrophages, granulocytes, and lymphocytes that are subject to the cytolytic effect of tuberculin. An electronmicroscopic check (Bassermann) revealed, as might have been expected, no qualitative differences of the tuberculin cytolysis compared with "normal" lytic processes. It did, however, show an acceleration of the process and a dependence on the degree of cellular sensitization; yet this (as shown by the works of, among others, Waksman et al.) has no strict relationship to the tuberculin susceptibility of the donor animal. Most authors assume that an antigen-antibody union underlies the tuberculin cytolytic reaction; and the work of Favour 1, of Miller and Favour, and of Miller, Favour, Wilson and Umbarger, makes it quite probable. This indicates the existence in the tuberculous plasma of a factor which is necessary for the occurrence of cytolytic tuberculin activity and seems to be gradually liberated from the lymphocytes. Cytolytic phenomena which correspond to those induced in experiments on animals have also been observed in man (Gangarosa, Inglefield, Thomas and Morgan). In these cases, the lysis by tuberculin of leucocytes from sensitized people did not run parallel to the skin reaction (J accard).

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However, no one has succeeded in finding a patho-physiological equivalent for the cytolytic processes. Investigations on liberation of lysozyme (Kerby and Chaudhuri), total metabolism (Marks and James), or oxygen consumption (Sanford, Favour, and Lindsey) of leucocytes from sensitized animals, provided no clarification of the toxic effect of tuberculin. From the diverse experimental material, we must draw the conclusion that even the simple allergic reaction represents a complex of superimposed antigen-antibody reactions; that these are complicated by constitutional and non-specific factors (Pepys), as, for example, Shwartzman's active substances (Packalen) or hormonal influences (Berdel and Wiedemann); but that, on the other hand, they themselves can give occasion for secondary, unspecific reactions (Schmid and Hagge). Thus sometimes the cellular, sometimes the humoral mechanisms come more to the fore (O'Neill). Further findings are probably to be expected, only if the individual hapten- (or antigen-) active components of tuberculin can be isolated and their behaviour tested in their relationship to the completely or partially hyperergic organism or cell system, as has been attempted by, for example, Fabrizio.

4. lmmunochemistry of the Tubercle Bacillus 1. PROTEINS In the United States and in Sweden, completed studies have led to the finding that the tubercle bacillus in the infected organism (Dennhardt; Funfack, Oberiick, Schmid, and Stoermer; Wildfiihr) and in artificial cultures forms a series of intrinsically quite inert proteins, besides other chemically defined substances. Of these proteins, the one presumably most important has a molecular weight of 32,000. After death of the bacteria or even during their multiplication, these proteins diffuse into the surrounding medium and, with cautious preparation (especially if higher degrees of temperature are avoided) can be obtained in crystalline form (Seibert1). These proteins can be obtained easily and in large quantity from unheated culture filtrates of tubercle bacilli, and in lesser amounts from the bacteria themselves (Raffel2, Toenniessen); in Germany they are designated as Tuberkulinmuttersubstanz (TMS, tuberculin mother-substance). In experimental animals, after parenteral administration, they induce a hypersensitivity; hence, they are total antigens. In sensitized animals, after intravenous re-injection of TMS, anaphylactic shock occurs; after intracutaneous injection, characteristic inflammatory skin reactions of the nature of the Arthus reaction, are said to arise (Enders; Lewis; Lewis and Seibert; Reichle and Goldblatt; Seibert2 ; Seibert, Pedersen and Tiselius; Smith). According to Seibert, the hypersensitivity reactions can be triggered not only by natural TMS, but also by preparations that have been subjected to the influence of strong physical or chemical agents, especially higher temperatures as in the case of the commercially familiar old-tuberculin. On the contrary, according to Seibert, a sensitization to TMS occurs neither with the fission

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products of TMS contained in heated culture filtrates (this particularly refers to a body with a molecular weight of 16,000) nor with the commercially familiar tuberculins obtained under heat action; thus, according to Seibert, they have only hapten characteristics. Nevertheless, from unpublished investigations of Prigge, it appears that weak sensitization is possible even with purified tuberculin GT (see below), while when using this preparation, a very much greater amount of active substance can be injected than when using Koch's old-tuberculin. On the other hand, the anaphylactic phenomena that can be induced by intravenous injection of highly concentrated tuberculin in TMS-sensitized animals are likewise only slight (Prigge 4). These observations can be explained more easily by the presence of a residual amount of antigen in the tuberculin than by the transformation of the antigen into a hapten. Furthermore, Magnus and Edwards, and Smith and Scott could not confirm Seibert's statement. Recently, residual antigenicity has actually been claimed for old-tuberculin (Kubo, Mori and Natori), and Murohashi and Maeda, as well as Cluff, have reported hyperergies of the "delayed" or of the "Arthus" type after repeated skin tests. The allergy of the tuberculously infected organism is intelligible on the basis of the concepts developed by Seibert, as a hypersensitivity induced by the proteins of tubercle bacilli, in particular by Tuberkulinmuttersubstanz; this hypersensitivity is operative not only against the total antigen (TMS), but also against those of its fragments with hapten characteristics, contained in the tuberculin. However seductive this idea may seem, it can nevertheless not be considered as fully proved. It has not yet been made clear whether TMS can induce a specific allergy in man against old-tuberculin in the same way as does a tuberculous infection. For the tuberculosis allergy is a predominantly cellular phenomenon which is only exceptionally accompanied by the appearance of a precipitating antibody demonstrable by the classic methods. On the contrary, in man and experimental animals, which have been treated with TMS or cautiously prepared fractions from tubercle bacilli, large quantities of antibodies are found in the blood, and these react with the antigen in vitro in a characteristic manner with formation of precipitate (Seibert2 •3 •5 , Seibert, Pedersen and Tiselius; Kashihara). Whether we should attribute to these facts a decisive significance in the status of the allergy, can be left undecided. Further qualitative and quantitative studies on the reaction potential of the serum of tuberculous animals and men against the proteins of tubercle bacilli, especially TMS, will probably lead to a more fundamental

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clarification here than have the studies made up to now on this question (Dreyer and Vollum; Pinner and Knowlton; Raffel 2 ; Seibert2 •3 ·i; and others), which have given negative or highly contradictory results. Only the more recent works of Raffel on the waxes of tubercle bacilli, which will be gone into again later on account of their significance, have achieved a certain advance here. Tuberculoproteins and their fractions have become, in recent years, the subject of thorough investigations in which, depending on the end in view, various approaches had to be adopted (Baldwin, Gilbert, Iland and Jones; Heckley and Watson; Jones, Larsen, Vardaman and Baisden). The tuberculoprotein fractions (TPA and TPT respectively) obtained from unheated filtrates of cultures of tubercle bacilli by ammonium sulphate and trichlor-acetic acid precipitation, were highly antigenic for guinea-pigs and rabbits; with repeated administration, they induced greater concentrations of precipitins, as well as anaphylaxis and Arthus phenomena. Hence if these substances-valuable for their high tuberculin activity-are to find application in diagnosis, we would have to destroy their antigenicity, which is generally easy to do by the action of heat. The product obtained by Seibert4 just recently from heated culture filtrates, containing added glycerine and phenol, and precipitated by trichloracetic acid, this so-called PPD, is tuberculin-active; yet, in the doses administered in practice, it is not antigenic (Cramb, Meier and Seibert; Tukey, Dufour and Seibert); hence, it has found wide application for diagnostic purposes, although the freedom from antigens of the preparation could not be established by all authors (see above). A significant antigenicity of PPD is in no case demonstrable; and that activity of tuberculin currently recognized by Boyden and Suter, which stimulates formation of heterologous antibodies, should fall rather within the province of Freund's adjuvants (Freund; Freund and McDermott) than within that of true antigenicity. In this connection, we must refer once more to the possibility already mentioned of making tuberculin completely antigenic by coupling it with "carrier" substances. This fact gains particular significance since Aoyama not only demonstrated the activating power of well-known heterologous substances (body-foreign proteins, aluminium hydroxide) but also recognized as activators of tuberculin homologous nuclear materials, especially depolymerized DNAs. Hence, we must take into account that nuclear materials liberated from homologous breakdown

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products, for example from tuberculous necrosis, can lead to antigen completion of tuberculin liberated in, or introduced into, the body. With the introduction of finer techniques of measurements (Prigge and Dohmen; Eissner1 •2) and on grounds of clinical observations, it appears that we do not always succeed in obtaining preparations with maintenance of uniform tuberculin activity (Holm and Lind). Here, the conditions of culture, among other things, play an important role (Herrmann). Physico-chemical investigations have also shown (Seibert, Pedersen and Tiselius; Seibert and Dufour 1) not only that PPD contains great quantities of impurities (nucleic acids and polysaccharides), but that the protein-fraction itself is by no means homogeneous. The PPD-S which was, in consequence, prepared by Seibert and Glenn by improved methods of purification (repeated ammonium-sulphate precipitation) is today used preferentially in Anglo-Saxon countries for diagnosis (cf. Seibert and Dufour2) ; it contains hardly any nucleic acids and only a small component of polysaccharides. Whether these represent an integrant component of the tuberculin-active substances (Herrmann) is still undecided. In Germany, even before the Second World War, the development of a purified tuberculin corresponding to PPD-S was begun by the Georg-Speyer-Haus (Prigge) in conjunction with the Hoechst Farbwerke (Lindner); this is marketed today as "GT Hoechst." It contains almost nothing but specific, non-antigenic protein derivatives of various molecular weights; and it leads to a slight sensitization only in very large doses not used in diagnostic applications for man. The non-uniformity of the protein fraction was further investigated by Seibert 9 in unheated culture filtrates, since the question is of great scientific and-in view of the possibility of an active vaccination with antigenic portions of tubercle bacilli-of practical significance also. In this work she could isolate, by fractionation of the complex tuberculoproteins with alcohol at low temperatures, and study for their biological characteristics, first three protein fractions, A, B, and C, also differentiable by electrophoresis, as well as two polysaccharide components, I and II, the percentile proportion of which depends on the strain of tubercle bacilli (Seibert, Soto-Figueroa and Dufour; Seibert, Crumb and Seibert). The nucleic acids and nucleoproteins that move rapidly in an electric field and are without tuberculin activity can be separated electrophoretically from the true nucleo-proteins (A-C) (Seibert6)-as can the immobile polysaccharides.

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The methods of preparation of Heckley and Watson, carried out very conservatively, led to similar fractionation results. Protein A showed in general the strongest tuberculin activity, this being presumably identical with the previously crystallized tuberculoprotein of Seibert; it has the molecular weight of 32,000 postulated by The Svedberg as the most frequently encountered unit of protein structure. The somewhat less active protein B-with a molecular weight of about 16,000-we can consider as a breakdown product of A; while C, which is insoluble in acid conditions (pH 3.8-4.7), shows a diverse activity (Seibert, Crumb and Dufour), and is distinguished from A by its UV spectrum, has turned out to have a high molecular weight. Recently, an additional glucoprotein, highly active biologically and rich in carbohydrates, was isolated from the bodies of tubercle bacilli by urea extraction, by Seibert, Soto-Figueroa and Dufour, and by Seibert and Fabrizio; this presented a certain physico-chemical similarity to the C protein from culture filtrates, yet had a stronger action on skin. Chemically, this glucoprotein is characterized by its particularly high content of alpha-epsilon-diamino pimelic acid, an amino acid that occurs very rarely in nature but characteristically in certain mycobacteria. Immunologically, the glucoprotein appears inactive in animal tests, yet active in tissue culture towards polymorphonuclear leucocytes of uninfected animals pretreated with the glucoprotein. It thus possesses antigenic properties and, in fact, presents a certain similarity to the C protein. As already mentioned, the allergic skin reaction of animals sensitized with proteins, occurs within the first hour after intracutaneous reinjection of the antigen, of ten after only a few minutes. We are here speaking of the "direct," "anaphylactic," "urticaria)" type of reaction, of an "immediate" reaction or of the Arthus phenomenon in the strict sense. The reaction appearing in tuberculous individuals after tuberculin injection, on the contrary, does not appear before 7-8 hours; it thus represents the "delayed" type of reaction, and we even speak of the "tuberculin" type of skin reaction. Seibert, Pedersen and Tiselius assume that no fundamental significance pertains to these distinctions, and that they depend only on the physical properties of the molecules inciting the reaction, so that in fact TMS always calls forth the urticaria), and tuberculin the delayed type of reaction. Yet Prigge3 could trigger with TMS, in tuberculous laboratory animals, always only the same late reaction as with tuberculin. Whether, conversely, we should include the skin reactions which

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we can produce with tuberculin in guinea-pigs sensitized by TMS among the urticaria! or (according to the findings of Seibert) delayed type, is undecided, since they frequently seem not to occur at all according to the investigation of Raffel 2 • These conditions, which need still further clarification, do not mean that tuberculous allergy can be taken exclusively as hypersensitivity to the proteins formed by the tubercle bacilli. Indeed lckert2 (inter alia, p. 454) even takes the position that the tuberculin reaction, or the course of reaction in Koch's basic experiment, "corresponds" to the Arthus phenomenon. Nevertheless, it must still appear questionable in the first place whether the proteins contained in the filtrates from cultures of tubercle bacilli, really play just the role that is attributed to them by Seibert. Bieling succeeded in producing a tuberculin preparation which was highly active in tuberculous animals, but contained neither protein nor protein breakdown products; we must, then, take into account the possibility that the protein and its derivatives prepared from the culture filtrates trigger the characteristic late reaction in animals allergic to tuberculins, because they contain the "essential" active substance in very small but highly reactive traces. This idea has also been discussed by Keller, and it gains a certain support from the fact that the whole range of tuberculoproteins thus physico-chemically distinguishable, possesses tuberculin activity. Of course, Seibert's investigations also show that, supposing it is present, this active substance must be very closely bound to the tuberculoproteins, and that it achieves its specific activity only through its union with them and only from a certain size of molecule on up (Keller). We possess no certain proof of the presence of prosthetic groups in the tuberculin-active protein molecules. Hence, Linder holds emphatically that particularly stable peptide groups, within the protein molecules, are the carriers of the tuberculin activity. The more recent investigations of Seibert and Kent, and of Seibert, Soto-Figueroa and Dufour, permit certain cautious assumptions about the correlation of biological activity with definite combinations of amino acids. Nevertheless, these developments are still entirely indeterminate, as are the investigations initiated by Prigge and Bockemiiller (unpublished) in the same field of inquiry, which have as object the preparation in pure form of the partial antigens of TMS, or of the derivatives contained in tuberculin.

2.

LIPOIDS

The lipoids synthesized from tubercle bacilli, have, in recent times, taken on an increasing significance. They are subdivided into several

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large, chemically defined groups: fats (acetone soluble), phosphatides (acetone insoluble), and so-called waxes (A: soluble in warm acetone, insoluble in cold; B: Anderson's "soft wax," soluble in methanolethanol mixtures; C: soluble in warm acetone, and also chemically similar to wax A; D: lipo-polysaccharides; Anderson 3 , Asselineau and Lederer3). Waxes are also the compounds that could be demonstrated (combined with proteins and carbohydrates) in the supporting materials remaining behind after intensive extraction of tubercle bacilli by Umezu and Wagner-Jauregg. Modern chromatographic methods (Philpot and Wells) also gave findings agreeing with Anderson's: the whole range of compounds extractable from killed tubercle bacilli with organic solvents, contained, in variable amounts, acetone-soluble fats, free mycolic acid, a mycocerosanic-acid-phthiocerol ester, as well as esters of mycolic and other fatty acids with polysaccharides, while the "acid-fast" fractions always contained free mycolic acid. By this sort of investigation-especially also those with the infrared spectrograph-of the lipoids in tubercle bacilli (Kubica, Randall and Smith), we can differentiate different bacterial strains; and the possibility also appears to exist of more closely defining biological variants by comparison of their lipoids, and of separating them from bacteriologically indistinguishable types. (a) Acetone-soluble fats These compounds are distinguished by the lack of steroids (Anderson, Schoenheimer, Crowder and Stodola) and the replacement of the glycerine, customarily contained in natural fats, by the disaccharide trehalose (Anderson and Newman). The acetone-soluble fats seem to play no role in immuno-biology. They may comprise up to 10 per cent of the bacterial dry weight. And besides saturated and unsaturated free fatty acids (Anderson 3), they contain neutral fats (Anderson and Newman), unsaponifiable substances and aromatic compounds (Aebi, Asselineau, and Lederer; Anderson and Newman; Stendal), as well as pigments and odoriferous materials (Kasuya). Anyhow, the pigment designated as phthiocol, which was isolated by Anderson and Newman from the acetone-soluble fat of human tubercle bacilli some time ago, has recently gained significance in problems of chemotherapy (cf. Prigge6). Numerous studies have been carried out to clarify its constitution, among others, those of Weygand and Schroeder, according to which, phthiocol represents a naphthoquinone derivative similar to vitamin K 1 • According to Toyama, it

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has the character of a growth substance for the multiplication of the tubercle bacilli. (b) Phosphatides The phosphatides formed by the tubercle bacillus have recently aroused great interest. These compounds, which contain only a little (about 1 per cent) nitrogen (Asselineau and Lederer 3), are identified by two fatty acids (tuberculo-stearic acid, and phthionic acid) which do not appear in other natural phosphatides. These two fatty acids are linked to a water-soluble body which can be broken down into glycerine and a phosphorylated polysaccharide. From this, Anderson, Lothrop, and Creighton, after dephosphorylating the polysaccharide, isolated a trisaccharide which seems to be composed of mannose and inositol. Thus, the actual building blocks of the phosphatides of tubercle bacilli are phosphatide acids with a high biological activity (Haas1), which are esterized with a carbohydrate and not with choline or ethanolamin (as in lecithin). The compounds contained in unheated and heated filtrates from bacterial cultures, display no pathogenic activity in normal animals. However, recent works of American, French, and Swiss authors very emphatically concur with the opinion expressed by Sternberg at the beginning of the century; this was that the phosphatide fraction obtained by fractional extraction of the tubercle bacilli with organic solvents was the carrier of the essential pathogenic activity of the tubercle bacillus, i.e., its tuberculogenic power, that is to say its ability to cause tuberculosis (Anderson 3 ; Fethke; Roulet and Bloch) . Anderson's phosphatide not only binds tuberculous complement in the presence of serum but-without protein linkage-incites the formation of antibodies (in its unpurified condition!) . So it must be a full antigen (Pangborn) . The tuberculo-stearic acid contained in the phosphatide, a laevorotatory, IO-methyl stearic acid (Spielman; Velick), appears to have no pathogenic effectiveness; whereas Roulet and Bloch were able to demonstrate a phosphatide acid as a biologically active component of the phosphatide fraction of human tubercle bacilli. This was specifically a compound of glycerine-phosphoric acid with a branching fatty acid not otherwise appearing in nature---C2 6H61COOH. Birch and Robinson held it to be an acid doubly substituted at the alpha-C atom , apparently something like an alpha-ethyl, alpha-decyl, tetradecylic acid. Phthionic acid has also been claimed as a 3, 13, 19, or alternatively a 10, 13, 19-trimethyl tricosanic acid (Polgar and Robinson; cf. also

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Cate! and Schmidt). The latter is said to possess, when prepared synthetically, all the chemical and biological properties of phthionic acid (Chaine), so that evidently this formula approaches very closely the actual facts. However, the configuration, indeed even the unitary nature, of phthionic acid is by no means conclusively clarified. Thus Ginger and Anderson have already been able to isolate from the neutral fat of human tubercle bacilli four different fatty acids with the empirical formula C24H4s02 up to C21H6402, of which the C2 6-phthionic acid corresponds with the one formulated by Anderson and Chargaff and described as active by Roulet and Bloch. Polgar 1 also, as well as Chanley and Polgar, prepared various dextro-rotatory fatty acids from the lipoids of human tubercle bacilli, with the empirical formula C 26 H 6202, which were alpha-beta-unsaturated; and they isolated a dextro-rotatory fatty acid designated as mycolipenic and a laevo-rotatory fatty acid designated as mycoceranic acid. Finally, Cason and Coad, and Cason and Sumrell demonstrated at least twelve partial components in a methyl-phthioate of Anderson's, the chain-lengths of which ran from C 23 to C 31 , and of which an alphabeta-unsaturated, methyl-branched, fatty acid with an empirical formula of C21H 6302, made up a quarter of the total. This compound, designated as phthienic acid, proved to be extraordinarily active biologically. The investigations carried out by Husseini and Elberg showed that phthienic acid, as well as the analogous saturated phthianic acid (the latter to a lesser degree), was tuberculogenic, producing epithelioid and giant cells in rabbits; which stands in a certain contradiction to the results of similar investigations with phthionic acid (Cate! and Schmidt). The concept advocated some years ago by Prigge6 and Prigge and Kiksch, according to which the phthionic-acid complex contains unsaturated components, seems henceforth conclusively confirmed. Bovine tubercle bacilli seem to form no phthionic acid (Roulet and Brenner); we may, however, presume that a similar compound is formed by them as the bearer of their tuberculogenic activity. The activity of the phosphatide acids studied by Roulet and Bloch, or of the phthionic acids characteristic for their structure, can be compared with the effects of the poisons of other bacterial agents in many respects; whereas for tuberculin, as we have seen, such an analogy does not hold. Of course, phthionic acid develops its full activity only in the complete molecule of the phosphatide acid. Roulet and Bloch explain this peculiar relationship by the circumstance that the complete

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molecule contains a hydrophobic paraffin chain and a hydrophilic glycerin-phosphoric-acid residue. To the former it owes its fat-solubility; to the latter, the capability of forming aqueous emulsions. It is more easily taken up by the cells, and disperses in them otherwise, than do the free fatty acids. The latter, which must represent the actual "poison," thereupon become free intracellularly as a result of a hydrolytic cleavage process. Of course, the works cited (Polgar; Chanley and Polgar; Cason and Coad; Cason and Sumrell, see above) give evidence that the tubercle bacillus forms not phthionic acid, but corresponding unsaturated "dehydro-phthionic acids" with higher direct activity, which transform into phthionic acid only on artificial saponification of the glyceride. This being so, it is immediately comprehensible why the phosphatide acid is more active than the phthionic acid prepared from it (Bibi. in Prigge6). According to Smithburn and Sabin2 , the phosphatide of tubercle bacilli evokes a stronger reaction in tuberculous animals than in normals; the objection raised by Holley, that this phenomenon, analogous to Koch's, might be explained by the presence of impurities containing proteins, is decisively refuted. Paraf and Desbordes have further reported that they have reproduced with synthetic, unsaturated acids of the type of phthionic acid, not only the tuberculogenic activity of Roulet and Bloch's phosphatide acid and of phthionic acid, but have also evoked an allergy in guinea-pigs by repeated injections of small doses; they also report that in the pretreated animals, an infection with living tubercle bacilli ran a course with all the characteristics of Koch's phenomenon. Conversely, the injection of a small amount of acid into tuberculous animals excited the appearance of a pronounced hyperergic reaction, likewise in the nature of a Koch phenomenon. Even in tuberculous human beings, a characteristic skin reaction could be induced with the acids under consideration (Cate! and Schmidt; Catel and Weidmann), whereas uninfected humans remained always reaction-free. Of course, Paraf, Desbordes, Buu Hoi, Ratsimamanga, and Cagniant later thoroughly confirmed the facts established by Para{ and Desbordes; these indicated as relatively slight the degree of the allergy in the animals pretreated with the organic acids being investigated, and the Koch phenomenon observed by them after infection with living tubercle bacilli, as attenuated. On the contrary, very characteristic allergic phenomena were always observed in primarily tuberculous animals after later injection of the acids. As the

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fatty acids by themselves induce no definite allergy, but excite pronounced allergic reactions in the tuberculous organism, a hapten character is ascribed to them (Catel and Schmidt). Buu Hoi and Jouin have attempted to make complete antigens of substances similar to phthionic acid by linking the carboxyl group to the free amino groups of egg albumin and other proteins, so far without results, however. Hence, it still remains unclear how one should regard the development of the allergy directed against the fatty acids under consideration. In no case should we assume that these fatty acids were, perhaps, built into the TMS molecule. For, neither TMS nor tuberculin evoke the tuberculogenic activities of the phosphatide acid demonstrated by Roulet and Bloch and of the phthionic acid. And on the other hand, the weight of the amounts of fatty acids necessary to initiate the reaction is very much higher than the weight of the necessary amount of tuberculin. The origins of the allergies in tuberculous patients, directed against tuberculin and directed against the phosphatide fraction of the tubercle bacillus, are evidently different in nature. (c) Waxes In recent times, considerable biological significance has been attributed to the wax fractions A-D (Anderson 3), which can be classified on the basis of chemical characteristics, and which Asselineau 8 divides into the alcohol-ether-soluble A-waxes contained in the crude phosphatides, and the chloroform-soluble waxes (B-D); this is particularly true as regards the establishment and form of expression of tuberculous allergy. The acetone-soluble A-waxes comprise, besides the biologically interesting hapten (Cate) and Weidman) mycolic acids (Asselineau1, and Asselineau and Lederer1), primarily true chemical waxes, among others phthiocerol, an aliphatic, monomethoxylated bivalent alcohol (Stodola and Anderson). The B-(soft-) waxes contain the already mentioned components, phthiocerol, fatty and mycolic acid esters, and glycerin (Asselineau 2), while Anderson's "purified waxes" (C+D) possess complex polysaccharides built up from galactose, mannose, arabinose, and inositol, besides glycerin and phthiocerol. Recently, Noll (J. Biol. Chem., 1957, 224, 149) succeeded in ascertaining the chemical structure of (D-free) C-wax by means of chromatography and infra-red spectrography. It is composed chiefly of a phthiocerol-dimycoceranate, a triglyceride of an unbranched fatty

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acid with 22-26 C-atoms, an alpha-monoglyceride of mycolic acid, and two fractions C 1 and D 1 not further identified. Asselineau and Lederer1 demonstrated in the "purified waxes" a lipo-polysaccharide fraction (wax D) . This consists of mycolic and fatty acids which are esterized with a phosphorylated polysaccharide containing galactose, mannose, and arabinose, and nitrogen-besides several peptide-linked amino acids (glutamic acid, alanine, and alphaepsilon-diamino pimelic acid)-whereas glycerine and phthiocerol are lacking in it (Lederer). Similar compounds have also been described by Haworth, and by Stacey, Kent, and Nassau. They resemble the lipo-polysaccharide "Pmko" isolated by Choucroun.1 These substances have a significant relation to the virulence of the strain from which they are obtained. In the mycolic acid, richly contained in most wax fractions but particularly in wax D, we are dealing with a weakly dextro-rotatory acid-corresponding, in its commonest form, to a beta-hydroxy acid of high molecular weight, with a long, aliphatic side-chain in the alpha position. It has the apparent empirical formula C 88 H 17604, and can be separated by chromatographic methods into an alpha- and a beta-mycolic acid. Of these, the alpha portion attains, in human strains, up to 90 per cent of the total mycolic acid (Lederer), which is generally linked, in the bacterial cell, with an acetone-insoluble lipo-polysaccharide of high molecular weight. It is, at least in part (Yegian and Vanderlinde), responsible for the acid-fastness of the mycobacteria, which becomes evident only when the carboxyl group of the mycolic acid is free (Asselineau and Lederer1 •2 ; Fethke and Anderson). Raffel 2 was able to induce a specific hyper-sensitivity towards tubercle bacilli and towards tuberculin, by means of a wax fraction isolated by him but apparently not protein-free (see below); whereas he found the TMS alone, as also the phosphatide fraction, inactive. However, Choucroun 2 succeeded in inducing a tuberculin allergy in normal animals even with a tuberculoprotein, if she injected the protein together with paraffin oil. In this connection, the works of Yamaguchi are also interesting; he drew the conclusion from electronmicroscopic pictures of dried distillates from tubercle bacilli, that paraffins or related substances must be present in them. Raffel and his school next attempted to show by thorough investigations that the wax fraction is responsible for the development of allergy of the delayed-reaction type, and even particularly the "purified wax" (lipo-polysaccharide), an ester of mycolic acid with poly-

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saccharides present in the tubercle bacillus according to Raffel's 5 latest publications. This form of allergy could be induced in guinea-pigs by injection of a tuberculoprotein-wax mixture in the absence of bacterial cells; whereas the tuberculoproteins alone evoked only an allergy of the anaphylactic type. The "purified wax," in a sense, conducts the tissue reaction-supplementing antibody formation and the development of anaphylaxis-in the direction of the delayed form of hypersensitivity (Raffel3). That there arises, in addition to the latter, a passively transferable anaphylaxis to the protein of tubercle bacilli, seems to be only an individual or gradational distinction from the reactions of tuberculous individuals. Even with antigenic substances foreign to tubercle bacilli, such as albumin-linked picryl chloride, or with egg albumin, a supplementary "delayed" allergy towards the corresponding antigen could be induced, which could be desensitized separately from the anaphylaxis (Raffel and Forney). The possibility of a simple accessory activity is discounted (Raffel, Arnaud, Dukes, and Huang). It remains to be seen whether the same mechanism accounts for the phenomenon already observed by Dienes and Schoenheit,-that egg-albumin, which otherwise acts only anaphylactogenically, evokes the "delayed type" of tuberculin allergy when injected into tuberculous foci. The fact already observed by Raffel3, and by Zinsser, Ward and Jennings, that fat-extracted tubercle bacilli do not sensitize, or only weakly, is now confirmed by Myrvik and Weiser, and referred to the reduction of the active wax fraction. It is well known that infectious allergy of the delayed-reaction type can be induced even by infectious agents other than tubercle bacilli (cf. Raffel 4 ). In tuberculosis, this reaction is made specific by the tuberculoprotein which becomes active along with the wax fraction (lipo-polysaccharide). Raffel 4 considers it as proved that allergy attained in this manner is identical with that of tuberculous infection. Even the cellular reaction, after injection of the lipo-polysaccharides, is similar to that arising in tuberculous infection (Suter and White); in it, there occurs a pronounced stimulation of the reticulo-endothelial system (White, Coons and Connolly). However, Pound has now presented findings which do not confirm Raffel's concepts. He was, indeed, able to confirm the antigenicity of this fraction-as did Choucroun previously, whose wax was presumably identical with that of Anderson (Takeda)-but not its specific activity as observed by Raffel. Thus, the wax he employed

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induced only skin reactions of the Arthus type, and the allergy obtained with it was passively transferrable with the serum of the sensitized experimental animal. Furthermore, it appears quite plain both from the biological work of Myrvik and Weiser, and from the chemical analyses of Asselineau and Lederer, that the waxes themselves must contain a protein component as an integrating element. Moreover, Raffel3 himself established that the antigenicity of the wax was destroyed by digestion with papain. Pound's findings are, however, insufficient to invalidate the results from the basic work of Raffel's school. Actually, Pound himself admits that as a result of the complexity of the lipoids in tubercle bacilli, wax fractions might well have been employed that were different in spite of similar methods of preparation. Nevertheless, from another direction (Platt), doubts have already been expressed that the wax fraction alone should be made responsible for the development of the tuberculin allergy that runs a course of late reaction; for this type of hypersensitivity can rise also in diseases whose agents are by no means distinguished by a high wax content (variola). And Gell and Hinde (on grounds of histological investigations on the Arthus phenomenon and its relations to other types of allergy) regard the delayed form of reaction as the first step in the cellular conversion in the reticulo-endothelial system, which is followed by total immunization with increased maturation of plasma cells and formation of humoral antibodies of the anaphylactic type. According to this, tuberculin allergy is to be regarded as a sort of "incomplete" immunization. Salvin advanced similar findings in sensitization researches on guineapigs, with diphtheria toxoid and egg-albumin. In these, there appeared first a "delayed allergy" (passively transferable with exudate cells) with no demonstration of circulating antibodies; on which followed an allergy of the Arthus type with demonstrable humoral antibodies, along with extension of the sensitization. Here there arise certain parallels to the concepts of the LatinAmerican authors on immunization by BCG vaccination (cf. chapter V). Recently, Bloch, Sorkin and Erlenmeyer, Noll and Bloch, and Asselineau, Bloch and Lederer, have isolated from tubercle bacilli a petrol-ether- and chloroform-soluble wax fraction designated as "cord factor"; and Spitznagel and Dubos have isolated a monochlorbenzolsoluble lipoid, not further identified chemically, and designated as "fraction 7." Both substances are intrinsically toxic, and seem to

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stand in close relation to the virulence (Bloch 1) and toxicity of the tubercle bacilli. The cord factor has since been more precisely analysed chemically (Asselineau, Bloch and Lederer; Lederer; cf. also Noll). It is a dimycolic-acid ester of trehalose, N-free in the highly purified state, with the empirical formula C 186H 366O11±10 CH2, and a molecular weight of 3000. Already, analogous compounds have been synthesized which were cord-active. Although the biological properties of this class of substances are still comprehensively obscure, they have already been involved in immuno-biological investigations; thus, Helmert attempted to obtain, by "decordization" and subsequent killing of virulent tubercle bacilli, a finely dispersed vaccine material which would be definitely active in immunization but non-toxic. The attempts at immunization undertaken with this on guinea-pigs, however, gave only results which could be obtained equally well with pathogens killed in the same way but not "decordized," or with BCG. As regards the allergizing activity of the toxic fractions, no investigations are as yet available. However, Bloch's purified cord-factor has been employed for immunization in sub-lethal doses (Lederer1), without, on the whole, any success in attaining a reliable immunizing effect. 3.

POLYSACCHARIDES

In the carbohydrate fraction that can be isolated from tubercle bacilli and their culture filtrates, compounds appear to be contained which also excite allergic reactions in tuberculous animals, yet are harmless in normals (White). Our knowledge of the properties of the polysaccharides of tubercle bacilli does not yet permit any adequately clear picture of their biological significance, in spite of numerous isolated investigations (Bloch and Roulet; Heidelberger and Menzel; Klopstock and Vercellone; Lindner and Oelrichs; Roulet and Brenner; Sandor; Takeda, Ohta and Sato; cf. also Kall6s and Kall6s-Deffner). Nevertheless, Ender's concept, according to which the anaphylactic (not the "delayed" hyperergic) reaction status in tuberculous animals is determined primarily by antigens containing polysaccharides, is at least not without basis. As regards the chemical structure of the carbohydrates obtained from tubercle bacilli or from their culture filtrates, at least a few definite advances have been achieved in recent years, which have led even to practical results. Certainly, the polysaccharides obtained by the use of various

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preparatory procedures and raw materials largely correspond as regards their elementary building-blocks (Haas1); nevertheless, evident structural differences occur between products isolated from the bacterial cells (Haworth, Kent and Stacey; Heidelberger and Menzel; and Menzel and Heidelberger), the "wax polysaccharides" (Anderson 2 ; Anderson, Peck and Creighton; Anderson, Reeves and Stodola; Haworth; Haworth, Kent and Stacey; Stacey 1 ; Suto-Nagy and Anderson) and, finally, the polysaccharides I and II obtained from filtrates of cultures of tubercle bacilli by Seibert and Watson 1 and by Seibert, Stacey and Kent. By analysing the building-blocks of the somatic polysaccharides of tubercle bacilli, one can demonstrate the presence of the most varied carbohydrates. Thus, Chargaff and Moore, and Kent and Stacey found a serologically inactive glycogen, corresponding to that obtained from animal tissues, in fat-free extracted avian and human tubercle bacilli. Further, Laidlaw and Dudley were able to isolate a reducing, serologically active polysaccharide which was composed of pentoses up to about 30 per cent, and was finally broken down into D-mannopyranose, L-rhamno-pyranose, D-arabo-furanose, and hexosamin, by Haworth, Kent, and Stacey. Anderson earlier succeeded in separating the carbohydrates linked up with the lipoids of tubercle bacilli-especially with the waxesinto D-mannose, D-galactose, and D-arabinose. The more recent works of Haworth, Kent and Stacey have also come to similar conclusions, according to which these carbohydrates represent complex, branched chains with units of D-galacto-pyranose, D-manno-pyranose, D-glucosamin, and D-arabo-furanose. Also from the cultures, filtrates of tubercle-bacilli, polysaccharides have already been isolated, by Millier, and by Dorset and Henley, which contained D-arabinose and D-mannose. However, not until the work of Seibert, 10 and of Seibert and Watson 2 , using electrophoretic methods, could the polysaccharides be precisely separated from the proteins and nucleoproteids. The products thus obtained consist of an invariable fraction containing 0.2 per cent nitrogen, mannose, galactose and arabinose, with a molecular weight of 9000, and of a fraction containing 0.8 per cent nitrogen, with a lower molecular weight. The first compound, now designated as polysaccharide I, is unusually active serologically. Polysaccharide II was finally isolated by Seibert, Stacey, and Kent: as of very high molecular weight (100,000-500,000), distinguished by its high glucose content, glucosamin, and the lack of arabinose; and appearing to be a lipo-polysaccharide in whose molecule one lipoid unit occurs per eleven carbohydrate units.

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With the exception of the high-molecular polysaccharide II, which is evidently a true antigen and leads to the formation of precipitins on injection into rabbits, we should regard as haptens the available isolated compounds of the carbohydrates mentioned. In recent times, the tuberculo-polysaccharides have attained, through the attempts to apply them for sero-diagnostic purposes, a significance that the protein derivatives had enjoyed for some time. Since Shaeffer's report on the presence not only of anti-tuberculoproteins but also of anti-polysaccharide antibodies, numerous investigations have been conducted on the serological activity of the polysaccharides of tubercle bacilli; these, however, in so far as they were expected to serve in diagnosis of human tuberculosis, turned out to be almost uniformly disappointing (Iland). Only with the introduction of the indirect haemagglutination and haemolysis methods (Keogh, North and Warburton) into tuberculosis research (Middlebrook and Dubos), and their tannin modification (Boyden), have the tuberculopolysaccharides gradually begun to play a greater role in immuno-biological research and in the clinic (Takeda; Takeda, Watanabe, Kasai, Aoki, Kuribayashi and Hoshino). The findings advanced by Middlebrook and Dubos, and confirmed and extended by Grabar, led Meynell to draw the conclusion, since generally accepted, that in Keogh's antibodies we are dealing fundamentally with antibodies directed against polysaccharides, in Boyden's antibodies, against proteins. Thus, it is possible, by means of adsorptive techniques, to separate mixtures of polysaccharide and protein antigen fractions, such as occur in PPD for example (Meynell), and identify their homologous antibodies by absorption. By the application of these methods (which are undoubtedly capable of further development) first results have now been achieved which in part confirm older concepts and in part have divulged new information. Thus Lamensans, Grabar, and Bretey isolated from tuberculin a protein fraction which affected the skin but was not haemo-sensitizing, and a polysaccharide fraction which had no effect on skin but sensitized erythrocytes. Boyden and Sorkin1. 3, and Sorkin and Boyden were able, by using the tannin method on unheated culture filtrates, to differentiate serologically several proteins obtained by chemical means, and, in addition, to demonstrate several non-sensitizing proteins. A correlation with Seibert's fractions A, B, and C, was, however, not possible with certainty-except for a preparation analogous to C; but several fractions could be isolated from PPD and other heated filtrates, and these were related serologically to each other and to the fraction analogous with C from the

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unheated filtrates. On the other hand, it was possible to demonstrate by the Middlebrook-Dubos technique the presence, in unheated filtrates, of at least two of the fractions, in heated filtrates, of one, evidently consisting of polysaccharides. Schiff too distinguished in GT a protein effective on the skin but inactive serologically, and a polysaccharide haemo-sensitizing but ineffective on the skin. The farther work is pressed in this direction, the more evident becomes the multiplicity of the substances formed by the tubercle bacilli and active in one or another connection, or of the antibodies evoked by them (Boyden and Sorkin2 •3 ; Rheins and Thurston; Thurston, Rheins and Huziwara). The attempts to attribute individual serological qualitites to definite chemical substances, are, of course, only in their infancy (Boyden and Sorkin2); here, the modern physico-chemical methods of electrophoresis (cf. Sorkin and Rhodes; Sorkin, Rhodes and Boyden) promise new information. The great difficulties confronting the identification of individual antigen fractions can be envisaged from the work of Pope on diphtheria toxin. Up to now, we are not in a position to make a statement as to whether the antibody demonstrated by any particular fraction of the filtrate from cultures of tubercle bacilli is directed against only a single antigen, or whether it represents a comprehensive complex which possesses more antibody potentials than can be demonstrated by the antigen employed in the case in question. Accordingly at present there still exists no possibility of demarcating the allergic phenomena evoked (or triggered) by entirely diverse compounds (proteins, phosphatides, waxes, carbohydrates) from each other with perfect distinctness. Developments are still in flux; but they indicate that the old doctrine of "partial antigens" still has its heuristic significance; and that it owes to modern investigations on the chemical structure of the tubercle bacillus, only a more precise formulation. In any case, the concepts that seek to interpret tuberculous allergy as hypersensitivity towards a single compound, can no longer be sustained.

5. The Relationships between Immunity and Allergy Currently, great difficulties still attend the linkage in a single explanation of such obviously diverse phenomena as immunity and hypersensitivity towards the tubercle bacillus and its corporate parts. The majority of the authors concerned with this problem advocate, on various grounds, the point of view that we are dealing with processes which ought not to be equated or whose fundamental interconnection must be considered as at least doubtful (Boquet and Negre; Calmette2 ; Ickert 1 ; Lange 4 ; Raffel2; Rich 2 ; et al.). In this regard, proponents of BCG vaccination must naturally express themselves with particular care (Wallgren7); for, in that procedure, the indication for vaccination is based simply and solely on the assumption that the pre-allergic anergy signifies lack of defence, but hyperergy, on the contrary, signifies immunity. Consequently, even the official publications of the World Health Organization (WHO-The Research Office, Copenhagen) advocate the point of view that immunity is a consequence of allergy. "The significance of tuberculin hyperergy as regards immunity against tuberculosis is still the object of violent controversies, since one school considers allergy and immunity as not interdependent and even perhaps as in opposition, while another considers them as closely connected. The heads of the WHO/UNICEF campaign for BCG vaccination have identified themselves with the latter concept; and they have adopted the opinion that in practical procedure, the conversion of the tuberculin reaction from 'negative' to 'positive' after vaccination indicates the acquisition of immunity." A view opposed to that mentioned at the outset, on the distinctiveness of allergy and immunity, is advocated by Bessau3, by Catel and

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Daelen1, by Hense1 2-1 , by Jordan, by Kall6s and Kall6s-Deffner, by Pottenger and Jensen (cited by lckert4 ) and by Prigge3 ; this is, that the immunity is only a consequence of the allergy and that this interpretation must be maintained also for the special conditions of tuberculous allergy. Berdel and Buddecke, within the framework of the theory developed by them, consider the hyperergy and immunity of the tuberculous organism as allergic step-reactions in a single continuous process, in the over-all course of which, however, the antigen of each stage is the product of the union of the antigen of the preceding stage with the corresponding antibody. Thus, "pathergy" (hyperergy) and "phylergy" (immunity) appear as: "causatively and temporally consecutive, and then contemporaneously existing, phenomenally evident, individual steps of activity in the evolving, total allergic potential." A careful analysis may well lead to the conclusion that the opposition to causal linkage of immunity with allergy is, for the most part, due to the fact that definitions have been drawn up with insufficient accuracy for the terms used. Without going into the arguments in detail, we will accordingly limit ourselves to an heuristically useful presentation of the relationships. It has already been mentioned that the organism becomes hypersensitive towards almost all specifically foreign proteins, even if these are previously quite neutral, after parenteral administration; and that it forms "antibodies" which are not only cellular but which also pass into the circulation and can be demonstrated in the blood serum. A reaction takes place between the antigens and the corresponding antibodies, not only in the living organism but also in vitro. The reactionproduct often makes itself evident by forming precipitates (Doerr2). It now appears that numerous bacterial and animal toxins, for example, diphtheria toxin, rattle-snake venom, etc., against which the mammalian organism can form protective substances, also are of protein nature (see for particulars Prigge1; Micheel; Pillimer, Wittler and Grossberg; et al.). These proteins too are precipitated specifically by their antibodies in vitro. Of course, there are no unequivocal observations that the pre-treatment with the toxic proteins leads to phenomena of hypersensitivity analogous with those from pre-treatment with primarily non-toxic proteins; on this account, the course of Riehm's antigen-antibody reactions in such cases are considered as certainly belonging in the field of immuno-biology, yet are designated as nonallergic. But it is not very likely that the phenomena under consideration

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would be of basically different natures. We must rather assume that the reaction between the toxins and the corresponding antibodies in the organism, does not run a stormy enough course to cause the appearance of severe manifestations; indeed, this is primarily so because the absolute quantities of toxin administered for experimental poisoning of immunized animals are likely to be, in general, far smaller than the doses employed to demonstrate hypersensitivity against nontoxic proteins; and this is further shown by the fact that for poisoning, the subcutaneous route of injection is generally favoured, and for demonstration of the allergic, especially the anaphylactic reaction, the intravenous route. As soon as adequate quantities of a toxic protein, for example, diphtheria toxin, are available in a chemically pure form, it will indubitably be demonstrable that immunized ("sensitized") animals succumb with the same violent symptoms after intravenous injection of equivalent doses, as do animals pre-treated with non-toxic proteins. Hence, it can be considered as quite probable that the process of detoxification of toxic proteins, depends on the same mechanism as that which conducts the union between non-toxic proteins and their antibodies; and that "detoxification," considered in this connection, is merely a biologically unusually significant side-effect which, of course, is achieved only when the main reaction does not run so fulminating a course that a severe injury or even the death of the "sensitized" animal supervenes. Accordingly, there is no reason for us to wish to separate the processes of detoxification of bacterial toxins, which are considered to be classic immunizing phenomena, from the field of allergic phenomena or, to put it more plainly, of those that can be referred to hypersensitivity; nor should we explain them in principle otherwise than we do, for example, the phenomena of protein anaphylaxis or Koch's phenomenon and the tuberculin sensitivity of tuberculous subjects; we should particularly remember the conditions obtaining after administration of very large quantities of tubercle bacilli, which were briefly noted in the first paragraph of chapter II I. In recent times, investigations have succeeded in reducing hyperergy to tuberculin to the vanishing point, without reducing the immunity to tuberculosis; and these have been increasingly adduced as an indication that here allergy and immunity are phenomena of different nature; Boquet and Negre advocated this point of view as early as 1926 (Birkhaug1 ; Derick; Pagel; Rothschild and co-workers; et al.). On the contrary, Willis, Woodruff, Kelly and Voldrich were able to demonstrate by animal experiments that a tuberculin desensitization of hyperergically immune guinea-pigs leads-after the positive skin

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reaction has disappeared-to a substantial decrease in the power of defence against a subsequent virulent infection. Obviously in this sort of research evaluation of the length of survival is far more informative as to the immunity status than findings from sections from (generally prematurely) sacrificed animals. In this connection, the works of South American investigators are of particular interest, especially those of deAssis, of Rosemberg, and of Silveira (cf. Rosemberg 3); they accomplish, in man, a desensitization with living bacteria (BCG), using the so-called "concurrent vaccination," and at the same time produce only slight and rapidly disappearing hyperergy towards tuberculin or none at all. There is undoubtedly a difference between desensitization with tuberculin and that with complete bacteria the antigen-spectrum of which is far more comprehensive than that of tuberculin. Of course, the visible effect is initially the same. Nevertheless, desensitization with bacteria will also bind and eliminate antibodies that have no connection with the tuberculin reaction itself. On the other hand, with tuberculin desensitization, tuberculin inhibitors apparently arise in the serum, which-when added to tuberculin-restrict its reaction capabilities in the hyperergic organism (Stroder and Greferath). Wells and Wylie1, Berdel and Buddecke, and Berdel, Rubner and Wiedmann also demonstrated, in addition to the cellular antibodies involved in the hyperergic tuberculin reaction, a second antibody, which was humorally transferable and restricted the tuberculin reaction (and cytolysis); the quantitative relationship of this to the allergic antibodies appears to exert a material influence on the degree of the tuberculin reaction. I watsuru and Matoba advance the fermentationally active "tulysin" as a further substance possibly restricting the tuberculin reaction in the sensitized organism. In contrast to the investigations cited above, Kall6s and Kall6sDeffner have made the very revealing statement that in these cases the hyperergy is not decreased but that the course of the reaction following administration of tuberculin is so greatly accelerated that detectable symptoms do not appear. This explanation, which particularly refutes the frequently cited research of Birkhaug1, and others, on the "dissociation" of the allergic and immunizing processes, is well supported by experimental investigations of Fernbach and of Fernbach and Weichsel. Catel and Daelen 1 seek, in a way similar to that of Kall6s and Kall6s-Deffner, to explain "immunity sensu strictiori, i.e. "toxin resistance" that finds clinical expression in a disappearance of

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local sensitivity to tuberculin, together with good general condition and high power of defence of the organism against tuberculous infection"; they claim that in such cases (e.g., those occurring in intravenous tuberculin therapy) the organism no longer requires the indirect procedure of neoformation of epithelioid cells, but that all cells have "learned" the capability of defence without reaction. Recent Japanese works (Ohara, Nakadawa and Ikehata) also indicate the unitary mechanism of allergy and immunity. The attempt repeatedly made to define immunity against tubercle bacilli as "complete areactivity" and to set the positive anergy as a distinct phenomenon in contrast to allergy and especially to hyperergy (Letterer; Lutterberg; Nagel; Weiland) is thus incompatible with factual circumstances. The work of Fourestier and Blaque-Belair2 also shows clearly that the "positive anergy" described, which was referred by Bessau 3 to tuberculin antibodies, by no means represents a areactivity; rather, the overwhelming majority of these tuberculously infected and immune "anergics" can be recognized as bacterially allergic with the aid of the BCG test (Fourestier; Ustvedt), or by elimination of injected killed pathogens (HenseF; cf. also Bernard, Cros-Decam and LeJoubioux; Mande, Gaud, Houel, Genz, Goujou and Babeau). This likely tallies also with the cases reported by Arbmann and by Scadding of actively tuberculous patients insensitive to tuberculin. In most cases, the possibility remains that the appearance of a "positive" anergy associated with the cure of the infection is simply an expression of the decline of the tuberculin allergy and in no way indicates an immunity but an outright decrease of the power of resistance towards exogenous infections. We must not confuse with the so-called positive anergy, the not infrequent anergy appearing preterminally and presumably acting in a true local manner, such as can evidently sometimes arise even with a poor general condition of the infected subject. Thus, after the First World War, a similar phenomenon was observed when German children (tuberculin-negative) were taken to Switzerland for rehabilitation and then, while there, showed positive tuberculin reactions. Here, evidently, the allergy of the poorly nourished children became patent with improvement of their general condition. After the Second World War, Schneider reported on returned POW'S who, immediately after their homecoming, reacted as tuberculin-negative yet, after a few weeks, with improvement of their physical condition, fell sick of tuberculosis, with reappearance of the tuberculin reaction. Here at

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the same time is revealed the ominous connection of the allergy with at least certain forms of tuberculosis (cf. also Schulz). Andenaes found among the tuberculin-negative adults of his research material 7 per cent who displayed a bacterial allergy towards BCG on the application of Rosenthal's multi-puncture procedure. From this, he drew the conclusion that the "tuberculin-negative" group includes also those who have lost (or never attained) their power of reaction against tuberculin, but have retained their bacterial allergy. Ellison too emphasizes that "secondary negative allergy" is to be distinguished from primary, since the former bestows immunity despite the lack of tuberculin reactivity, though this is certainly not confirmed by Lunn's observations. The investigations of Ameuille and Canetti, of Canetti, of Saenz and Canetti, and of Miadonna show that a sort of latency can appear in tuberculin anergy; according to them, the allergy reappears with a substantial shortening of the pre-allergic phase, as soon as a reinfection has taken place. In the unusual types of reaction here described (in so far as their divergent course is not conditioned by physico-chemical, hormonal, immunological, or therapeutic influences) we are undoubtedly dealing with individuals who have responded negatively or inadequately to the partial haptens contained in tuberculin; on the other hand, they have reacted to the antigens that are not contained in tuberculin but certainly in the bacterial body, with formation of antibodies and development of a specific immunity. Fourestier's concept, which is also advocated by Chaussinand, 2 according to which bacterial allergy (in contrast to tuberculin allergy) may possibly induce specific immunity directly, is supported by the work of Feldberg, of Kraus and Dvorak, and of Steiner and Wigniolle. A true, i.e. bacterial-inclusive, anergy seems to be very rare (de Assis; Fourestier). In such cases, simply on account of the difficulties of assessing the true immunological status, one should be cautious and consider also the possible presence of an unusually high resistance. That the powers of defence dependent on resistance can be greater in some circumstances than those bestowed by immunity, has already been emphasized by Lurie (cited by Diehl). Hence, it has not yet been made clear whether "positive" tuberculin anergy is to be considered the ideal immunological condition as assumed by Bessau3 , by Fourestier, by Giraud (cited by Haase), by Saye, by Pottenger, by Schoop, and by the majority of the Latin American authors (de Assis;

THE RELATIONSHIPS BETWEEN ALLERGY AND IMMUNITY

47

Domingo; Rosemberg1. 2 ; Silveira2 ). Its mechanism can be explained readily in terms of the considerations presented by Kall6s and Kall6sDeffner, and by Catel. As is clear from the foregoing, an explanation of the hypersensitivity and immunity reactions towards tubercle bacilli, doing justice to the complex relationships, is impossible as long as one fixes one's attention only on reaction to tuberculin-as usually is the case. If one tries to bridge the differences that apparently exist between allergy and immunity, one must above all take into account information gained in recent years: that the organism apparently becomes hypersensitive towards a substantial number of the antigens formed by the tubercle bacillus. A better understanding of interrelations can be achieved only if one tries to explain "immunity" through the interplay of the reactions against all antigens of the tubercle bacillus or, at least, against the pathogenically important antigens. E. Jensen has recently called attention again to this point. Immunity against tubercle bacilli must be equated, not with hypersensitivity against one antigen, but with the hyperergy against all the antigens in question. Even the striking peculiarities, among which -in specific clinical pictures, for example, in scrofula (Joppich)hypersensitivity towards tuberculin is outstanding, should not be considered as arguments against a close relationship between hyperergy and immunity; but we should perceive in these only the expression of an unequal development of the various "partial" hyperergies and, hence, of the temporary or persistent dominance of the defence reaction against whatever antigen, or hapten, of the tubercle bacillus is the active agent in tuberculin. In this connection, one should also be clear that there is insufficient information as to whether the chief antigens (probably formed only in small quantities) are really to be found among the chemical buildingblocks recognized up to now; as to how far compounds obtained by the most varied methods should really be considered as identical with the natural substances built up by the tubercle bacilli; and finally, supposing this is really the case, as to whether they still possess antigenic activity even outside their original cell structure and in isolated condition. Lederer has just recently drawn attention again to the more than questionable identity of the tubercle-bacillus lipoids synthesized in vivo and those prepared by chemical procedures in vitro. Finally, even the question as to whether the noxious substances active in tuberculogenesis can really be classified collectively as antigens, appears not to have been completely and adequately clarified.

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The possibility remains open that compounds could occur in the phosphatide fraction, with no more ability to induce an allergy than have alkaloids, ethyl alcohol, mercury, or numerous other organic and inorganic poisons. However, the results obtained with alpha-alphadisubstituted fatty acids, already mentioned, contradict any such concept.

6. Methods of Vaccination 1. VACCINATION WITH FULLY VIRULENT TUBERCLE BACILLI

The knowledge that the already tuberculous organism reacts differently on tubercle bacilli invading from without than does the normal body, led, even in early times, directly to the idea of artificially inducing inactive infections; these would supposedly run so mild a course that they would actually provoke no recognizable symptoms of disease but, nevertheless, strong defence reactions, and afford the body a reliable protection, an immunity against exogenous neoinfection (von Behring; Koch; Romer). Of course, one does not readily achieve experimental conditions under which the desired effect can be actually demonstrated. The guinea-pig certainly shows great individual variation in its reaction towards virulent tubercle bacilli; yet it regularly undergoes a progressive, fatally terminating sickness, apparently even after infection with a single fully virulent germ. Similar conditions prevail with monkeys. On the contrary, with rabbits an experimentally induced tuberculosis will come to a stasis and recovery, at least in a portion of the cases, if only the virulence of the culture used in the infection is not very high. In sheep and cattle this is even the rule, as long as no massive injections are used. In man, we observe, according to the individual power of resistance and the degree of the infection, all grades between sicknesses continuing without symptoms and very severe ones running a fulminating course. Nevertheless, the possibility of artificially producing a lasting inactive or latent tuberculosis is there in principle. After Webb and Gilbert, and Webb and Williams had, on the basis of numerous animal experiments, gained the impression that one can achieve defence against more powerful infections, by means of very

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small, intrinsically harmless quantities of bacteria, they ventured to attempt, several decades ago, to immunize children with living, unattenuated tubercle bacilli. This procedure, proposed by the American authors, has been almost universally rejected, however, on account of its danger; even their research results have been called into doubt on grounds of various control experiments (Brown, Heise and Petroff; Bruyant). The attempt to create an immunity in man against natural infection by rubbing live tubercle bacilli into the skin (Bohme; Moeller) has led to no more significant results. The method is not only dangerous, but has also lost its theoretical basis since Rodiger2 has dispelled the widespread belief in the special immuno-biological status of the skin in the course of tuberculosis; he demonstrated that neither the doctrine of the rare occurrence of pulmonary tuberculosis in lupus, nor the notion that a skin tuberculosis gives protection against more severe internal tuberculosis, stands up to facts. In conclusion, it may be further mentioned that the vaccination of cattle with fully virulent bacteria, attempted by Romer certainly led to good protective results, but endangered the health of the animals too greatly and, so, had to be given up.

2.

VACCINATION WITH ATTENUATED TUBERCLE BACILLI

(a) Early experiments

As is well known, we distinguish several types of tubercle bacilli, of which only the Typus humanus and the Typus bovinus are of significance for man, while the murine and avian types and those for coldblooded animals play no pathogenic role. Nevertheless, the differences in the pathogenicity of the types for the various species of animals form the point of departure for numerous experiments in vaccination, especially of cattle with tubercle bacilli of the human type, which are only slightly pathogenic for them (von Behring2 ; Klimmer; Koch, Schiltz, Neufeld, and Miessner; et al.), and of man with attenuated bovine pathogens (Calmette and Guerin 1) or with the murine type, the so-called Vole bacillus (Wells 1). In laboratory investigation, the principle at the basis of these experiments led to promising results, thanks to the immunological relationship between the various types of mammalian tubercle bacilli; in practice, however, it has proved questionable. Even control experiments on the activity of the bovine vaccine material ("bovovaccin" or "tauruman") developed by von Behring and by Koch, which con-

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51

tained human tubercle bacilli attenuated by drying or by passage through glycerine broth, led to quite unreliable and, under natural conditions, usually negative results, as did other similar vaccination attempts based on the same principles. (Bibliography in Lange 3 ; Spiess3.) Since, furthermore, the possibility always existed of propagating virulent bacteria of the human type even with these methods (Calmette 1 ; Kosse!), they were soon discarded again. From early times, we have a very large number of publications on various experiments in immunization against tuberculosis with attenuated or hetero-specific pathogens. We shall not go into them individually here (cf. Lange), since no method useful in practice has developed from any of these experimental works. Let us only mention here the works of Arloing, of Baumgarten, of Dubos, Pierce and Schaefer, of Graub, of Hamburger, of Heymans, of Jotten and Pfannenstiel, of Kall6s, of Klemperer, of Kraus and Volk, of Lange and Lydtin, of Pearson and Gilliland, of Rossignol and Vallee, of Raw, of Smith, of Thomasson, of Webb and Williams, and of Zschokke and Saxer, whose methods not infrequently approached the BCGprocedure. They are extensively treated in the already mentioned publications of Calmette1 , Lange 3, and Spiess3• Even the comprehensive investigations carried out by Uhlenhuth, Miller, Grethmann and Hillenbrandt for prophylactic vaccination of cattle, using a bovine tubercle bacillus attenuated by age ("TB 18"), still only weakly virulent for guinea-pigs but tolerated by cattle in large quantities, led to no noteworthy immunity against virulent infections under natural conditions in the infected stable. The fundamental knowledge gained by Uhlenhuth and his co-workers possesses the greatest theoretical significance even today. Selter1 undertook experiments to induce a defence against severe infections in guinea-pigs and calves, with minimal quantities of bovine tubercle bacilli, primarily in view of the possibility of a practical application to man. In these, he employed a vaccine material designated as "Vitaltuberkulin," which contained bacteria that were living but attenuated in virulence. The bacterial material used for preparation of the vaccine was ground in an agate mortar until the majority of the germs were broken down; hence the preparation comprised a mixture of living and killed germs or fragments. Seiter advocated, in a particularly pregnant form, the opinion shared by many authors, that only a truly persistent infection, attenuated but leading to tuberculous tissue changes, is able to effect immunity; however, he considers it possible to conduct the protective infection with attenuated pathogens

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in such a way that it remains latent. According to Lange', the cultures used by Seiter can certainly not be considered sufficiently attenuated, since they themselves generally give rise to a progressive disease. Likewise, the findings achieved in the United States with the human strain "Trudeau," rendered of weak virulence by many years' culture on laboratory nutrients, can obviously not be transferred to practice because the virulence of the strain could not be adequately fixed at the desired level (Baldwin and Gardner; Krause and Willis). The same is said to be the case with the formerly virulent, but now allegedly nonpathogenic, diffusely growing strain of tubercle bacilli "Arloing," which has been cultured for more than fifty years on artificial media, and on which Gillissen and Rocholl have recently made a new report. We shall not go into the attempts to immunize against virulent pathogens with acid-fast saprophytes (Boquet and Negre; Mayer; Seiter, cited by Uhlenhuth 2) especially with timothy-grass bacteria or with Friedmann's so-called "turtle tubercle bacilli"; they were practically all without result. A report has been made again in very recent times of immunization findings with acid-fast saprophytes (Zebrowski), and the author goes so far as to draw a hypothetical deduction that so-called innate resistance against tuberculosis might possibly be a result of early immunization by (ubiquitous) acid-fast saprophytes; nevertheless, we can probably expect little new stimulus for tuberculosis vaccination from this direction. On the contrary, the pathogen of rodent tuberculosis, the Vole bacillus discovered by Wells' in 1937, a tubercle bacillus belonging to to the murine type (Brooke; Wells2), has recently achieved increasing significance. The reasons for this will be considered in sequel to BCG vaccination. The problem that arises in preventive immunization with attenuated tubercle bacilli can perhaps be most clearly formulated as follows: the protective infection must, on the one hand, be active (i.e. virulent) enough, to guarantee an adequate reaction of the organism against the immunologically significant antigens but, should, on the other hand, run a course such as to induce no manifest sickness. The span left between inefficacy and the danger of disease seems, however, to be but narrow in immunization against tuberculosis undertaken with living pathogens. (b) BCG A substantial forward step with regard to attenuation of virulence and with particular regard to fixation of the lowered degree of virulence

METHODS OF VACCINATION

53

was achieved by Calmette and Guerin 2 • The germ, designated as "BCG" (Bacille bilie Calmette-Guerin), was considered to be primitively a bovine strain of tubercle bacilli (cf. Grumbach 2), attenuated by passage over a period of thirteen years through artificial nutrients (glycerine-potato with addition of ox bile); its virulence has been so far reduced, that in general we can induce no progressive tubercular changes with it in guinea-pigs, but only localized effects. Innumerable animal experiments have been undertaken regarding the pathogenicity of BCG, which indicate that any recovery of its pathogenic qualities must be considered as highly unlikely. Even in human beings who have been inoculated with the customary doses of 0.05-0.1 (0.2) mg. parenterally or, especially, intracutaneously (the formerly prescribed oral administration is today almost universally abandoned by us, on account of its uncertain efficacy), a rise in virulence has been observed so far only in exceptional cases. It has long been established that the vaccinated children who perished in the Lubeck disaster were inoculated not with BCG but-as the result of an accidental interchange-with virulent tubercle bacilli (Lange and Pescatori). Nevertheless, BCG cannot rate as "virus fixe." This fact, universally recognized today, naturally introduces an uncertainty into BCG research. Quite notable differences have been established between the vaccine materials employed in different laboratories (B4>e; van Deinse3 ; WHO-Mon. Ser 12) which can certainly be traced only in part to the various methods of production (fresh, frozen, or lyophilized vaccine material: Birkhaug6 ; addition of glucose: Birkhaug4 ; lyophilization: van Deinse 1 ; B~e; van Deinse and Senechal; Ranganathan; Obayashi, Noguchi and Takano; adsorption on aluminum hydroxide: Fritzsche; addition of Tween-80: Dubos and Fenner; North and Newman; Obayashi and co-workers; cf. also van Deinse3 and Haas2 , as well as addition of various adjuvants: Cho and Obayashi; Cho, Obayashi, Iwasaku and Kawasaki). Undoubtedly, besides biological peculiarities of the strain in question, the proportion of living and dead bacteria (in part influenced by the technical processes mentioned) in the individual batches of vaccine material has a great influence on the allergizing effectiveness of a BCG vaccine (Berger; Meyer and Palmer); killed pathogens induce a distinctly lesser tuberculin allergy than living germs that establish a latent infection. On behalf of both bacteriology and chemistry, second thoughts have been expressed against the assumption of BCG stability. Thus

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Suter and Dubos were able to differentiate various strains of BCG by their different content of bacilli which develop into "cord-forming," "intermediate," and "non-cord-forming" colonies. The ability to form cords is alleged to run parallel, within certain limits, to the virulence of the respective strains so that, according to the proportion of bacterial variants in the vaccine material, a greater or lesser virulence is indicated. It is, certainly, a singular fact that it has been possible to isolate from BCG the toxic cord-factor which is apparently characteristic of virulence (Bloch, Sorkin, Erlenmeyer and Noll). Especially revealing on the question of BCG stability is the pronouncement of the chief of the BCG laboratory of the Pasteur Institute in Paris, van Deinse2 ; he explains in his official capacity that the Pasteur Institute declined responsibility for the serious vaccination complications observed in the Netherlands, since the Utrecht BCG laboratory employed not the Paris but the Copenhagen strain. Nevertheless, the Copenhagen strain undoubtedly goes back to Calmette's culture, just as do all other BCG strains. The divergence of conditions of culture adduced by van Deinse3 in explanation may be cogent in individual cases; nevertheless, as regards the problem of stability of the vaccine material, it must seem worthy of second thought if its virulence can be altered by a change in the nutrient medium. Now, naturally, changes in virulence need by no means always be augmentations of virulence; rather, contrary to Calmette's opinion, it has turned out that the average virulence of the strain is currently declining further (Hertzberg; Jacox and Meade; Wallgren4 ), especially when unfavourable nutrient media are used; the degree of attenuation seems to vary with the strains maintained in the various laboratories. All these factors of uncertainty have led to the demand for a "standardization" of BCG vaccine material (Aronson and Schneider; Sansonnens). Here, indeed, we must always keep in mind that in this connection we can engage only in efforts to obtain a "normalized" product prepared with the greatest possible uniformity; there can be no question of a true standardization in Paul Ehrlich's sense, as long as exact measurements of activity against an invariable standard preparation of constant (defined) activity are not possible. Of course, in discussing a general regression of residual virulence, we must not fail to mention that augmentations of virulence are not so completely excluded as might appear from a cursory survey of the relevant literature. Ramon2, among others, has recently called attention to this incisively.

METHODS OF VACCINATION

55

Besides the basic investigations of Nobel and Sole and of Chiari, Nobel, and Sole, whose findings are incompatible with the notion that BCG is absolutely harmless, we should mention the works of Feldmann, of Heymans, of Hutyra, of Lowenstein1, of Petroff 1 •2, of Pirquet, of Seiffert, of Uhlenhuth 3 , and of Watson, McIntosh and Konst; their conclusions, except for the publication of Petroff discussed by Neufeld, would not be very easy to disregard. In very rare but carefully confirmed cases, Kuster (unpublished) in Prigge's laboratory, has been able, by simple bacteriological means, to obtain from BCG aberrant subcultures of higher virulence and Beller has been able to re-establish the virulence of BCG in a quite considerable number of cases by inoculation of BCG cultures into guinea-pigs that had served in investigations on foot-and-mouth-disease virus; in fact, virulence was restored not only for guinea-pigs but, in accordance with the fact that the agent of vaccination belongs to the typus bovinus, also for rabbits; though, against the conclusiveness of Belier's experiments, objections have been raised, especially by Kathe (personal communication). However, it is by no means only research using animal experiments which shakes the thesis of the absolute harmlessness of BCG vaccination but, in increasing degree, clinical observations also. In this connection, we must keep in view that the number of complications-when we are not dealing with outright fatal cases-is, in its full extent, far beyond our grasp. The most diverse percentages are given for regional glandular reactions, abscesses, and ulcerations. We shall not go into these here, since they are mostly a matter of side-effects which, though tiresome and unpleasant, are after all harmless, and which we would gladly accept in the bargain for the achievement of an effective defence against tuberculosis (cf. Baumann4). Those pronounced organic alterations after BCG vaccination, of which the tuberculous origin could be confirmed positively, and which represent a gradual transition to the fatal progressions discussed below (cf. also James), have already presented rather more serious food for thought. Pulmonary changes which were interpreted as "hilitis Becegeica" were seen by Bahar in 63 per cent of cases after BCG vaccination. Pulmonary infiltration and enlargement of the hilar glands were also seen by Fourestier, Bidermann and Ettedgni, by James, by Melletier, and by Richards and Steingold, among others, in connection with BCG vaccination; whereas Wallgren 6 was never able to establish "BCGitides" after intracutaneous injection.

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But we have reports not only on pulmonary phenomena, but also on those in the eyes (Anderson; Frandsen), and skin (Kalkoff; Schreus and Doerner), and particularly several cases of lupus (Jensen 2 ; Jensen and Fridmont-Mcpller; Marcussen; and Blumenthal; Masek; Davies; Merkelen; Maire (this group cited from Zebrowski): and Bruyne; Odegaard; Gilje; Lomholt (this group cited from van Deinse4) and others). Furthermore, K. A. Jensen has called particular attention to the pathogenically important fact that the virulence of BCG is only insignificantly less than that of the pathogen isolated from lupus tissues. Conclusions have been drawn only with great hesitation from the incidents so far published, of which those cited represent only a small segment. As late as 1949, Wallgren4, one of the most distinguished experts on BCG, was able to assert: "Practically world-wide investigations indicate that the BCG bacilli are unable to induce any progressive tuberculous disease, either in man or in the usual laboratory animals* ... , so that I do not consider it necessary to linger further over this question." In 1955, the same author8 posed the question based on comprehensive considerations, "Should mass vaccination with BCG be discontinued in Scandinavia?" and answered in the affirmative. The occasion for this radical about-face was given, among other things, by a series of incidents, reported in recent times, which demanded grave consideration, and the connection of which with BCG vaccination is so far beyond all doubt that the question of the vaccine's harmlessness must be put to renewed test. Indeed, it is now twenty-five years since Nohlen and Sarvan reported the first case of death from BCG in a large laboratory animal, a monkey, though BCG is reputed to be as harmless for this species as for man. Since then, we have descriptions of a series of metastatic tuberculous foci (Beyer), of generalization, and of fatal sicknesses in the course of or subsequent to BCG preventive vaccination (Ariztia, Orellano and Del Rio, cited by Irvine; Despierres and Viallier; Domingo; Hollstrom and Hard; lmerslund and Jonsen; Irvine; Lopez-Vallejo; Minsouni; Svenkerud). If indirect consequences of BCG vaccination are taken into account (Dufour and Despierres; Price; Wolk; Ruziczka) which *That golden hamsters consistently succumb to a BCG infection (Hauduroy and Rosset) could not, on the other hand, be confirmed (Berger and Puntigam; van Deinse 3 ; Helmert, unpublished). According to Saenz, the death of the animals is to be attributed to the toxic action of the metabolic products of the pathogens.

METHODS OF VACCINATION

57

are not infrequently of allergic nature (Flesh-Thebesius and Lauche; Rotter and Vogt; et al.) then the number of BCG-complications is greatly increased. Various attempts have been made, most recently by Calwell, to discredit the causal relationship between vaccination and fatal termination in the cases mentioned; and it is certainly quite right that the relationship cannot be demonstrated with certainty in every case. All the greater, then, is the significance of cases of fatally terminating generalization, very recently reported by Meyer and Jensen, by Thrap-Meyer, Oeding, Hesselberg and Waaler, and by Falkmer, Lind and Ploman; these were so thoroughly worked over clinically, pathologically, and bacteriologically, that no doubts are possible or have been expressed on any part, as to the causal relationship between the fatal course and the preceding vaccination. On the contrary; even Wallgren, who is cited in all the writings propagandizing for BCG prophylactic vaccination, as the champion and advocate of the procedure, objectively deduces consequences from the events: "The knowledge that progressive BCG illnesses of this sort inevitably arise shakes our belief in the assumed harmlessness of the BCG bacilli. It is no longer possible to maintain that BCG vaccination is always innocuous. Up to now, we have taken the stand that as many as possible should undergo BCG vaccination, even in the absence of an apparent risk of exposure. We no longer have the right to include the innocuous nature of the vaccine among our arguments" (Wallgren8 •9). The number of fatal generalizations after BCG vaccination reported from Scandinavia was correctly designated by Wallgren' as "minimum." Hence, it should not be taken as proportional for the great number of those vaccinated throughout the world, in order to demonstrate the slightness of hazard involved in vaccination. From this, a complete repudiation of BCG vaccination by no means follows; it signifies only that the hazard connected therewith should be recognized and brought into a sensible proportion with the expected immunological effect. Wallgren8 , in consequence, today frankly advocates that in countries with a level of resistance and a morbidity similar to those in Scandinavia, no further mass vaccinations should be carried out, since the hazard of exposure in the dangerous years of early childhood is less than the hazard in vaccination (cf. also Baumann3). On the other hand, in regions and among population groups with a very high exposure hazard (undeveloped districts, heavily exposed groups, small children in tuberculous surroundings)

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individual BCG vaccinations should be undertaken now as formerly, since here the hazard of exposure exceeds the hazard in vaccination. The basic cause of the disturbing BCG incidents is only partly and hypothetically clarified. Thus, Wallgren 8 supposes that "possibly" an agamma-globulinemia might be basically implicated, a supposition that can confidentially be excluded at least for the case reported by Imerslund and Jonsen. Direct increases in virulence of the materials employed in vaccination can be excluded-at any rate, for the greater part of the episodes. The basic cause is clearly to be sought primarily in the milieu, i.e., in the vaccinated organism, the abnormally weak resistance of which makes possible an unrestricted spread of the inoculated germs. A further sidelight on this problem is probably given by the recently published reports of Cross and of Engelbreth, Holm and Eskelund, on cases of prolonged, abscess-forming regional adenitis following BCG vaccinations that were administered in the incubation period of measles or during an influenza. We may assume that the tuberculin allergy well known to be frequently associated with measles, played a certain part in the cases described. This is a further-clinical-indication of the close correlation between allergy and immunity. Here also belongs Belier's observation on the pathogenicity of BCG for guinea-pigs that had been treated, before the infection, with foot-and-mouth disease vaccine. Whether in this case we are dealing with an enfeebled resistance resulting from leucopenia, as Drager assumes for erysipelas infections after virus diseases (or vaccinations), will not be discussed here. Van Deinse4, as regards the observed "rise in virulence" of BCG, also weighs the possibility " ... that we have to do here with a still unknown mycobacterium closely related to BCG; or one might postulate a virus infection, as a result of which the BCG bacilli injected during vaccination could find a favourable nutritional medium in the necrotic gland substance .... " Nevertheless, the BCG pathogenesis seems to be beyond all doubt in those cases that were investigated with such bacteriological thoroughness, as by lmerslund and Jonsen (after Svenkerud) or by Thrap-Meyer (after Oeding, Hessel berg and Waaler). All these investigations testify that the virulence of BCG depends not only on the properties of the culture, but also on the nature of the terrain on which it develops and that under special circumstances, one must count with variations in virulence in the sense of an increase. We do not have sufficiently conclusive experiments to indicate whether

METHODS OF VACCINATION

59

BCG behaves in the extremely undernourished organism the same as it does under normal conditions; thus, before administering the vaccination to an inadequately nourished population, a painstaking survey of prevailing conditions would be required. In any case, a progressive tuberculosis can be induced experimentally with BCG in silicotic and also in undernourished animals (Myers) . Certain safeguards can be introduced simply in the process of preparing the vaccine material. We do not possess reliable knowledge as to how the attenuation of virulence in BCG has come about. But we might best picture the process to ourselves in the same way as Jensen 1 and Frimodt-Ms;,:lller conceived the transformation of cultures of tubercle bacilli from the (virulent) "S" into the (non-virulent) "R" form (cf. also Grumbach 2), or as Luers did the establishment of resistant bacterial strains; according to this, the non-virulent bacteria arise through mutation and gradually achieve a majority by a process of selection. Conversely, however, the results of Kiister's investigations admit the supposition, now also taken up again by Suter and Dubos, that the usual laboratory stains of BCG are not entirely homogeneous, but still contain scattered germs of higher virulence. On these grounds, only such vaccine sources should be employed in the preparation of vaccine material for actual use as have been obtained by micromanipulation from single-cell cultures of BCG. If tests of such cultures in animal experiments establish their complete harmlessness, then we hardly have to reckon further with the appearance of virulent variants as a result of dissociation processes; the possibility that virulent varieties might reappear from non-virulent is certainly present in theory, but is extremely improbable. • In Germany, the strains (cultures) used for preparing BCG vaccines have been subjected to government control* for many years and are periodically re-tested. Also from every single batch issued for purposes of vaccination, a sample must be sent in to the testing institute, to be held for six months as stock for supplementary controls. On the problems arising in connection with government control, its facilities, possibilities, and limitations, Eissner3 and Simon have commented at length, the latter especially for mid-Germany where, through Kathe 1- 3 , the facilities for control of vaccines and for the conduct of mass vaccinations have been set up. After what has been said above, it is obvious that under state *Conformably with RdErl.d. RMdl. of the 15th of January 1945-Ag.7670/44/ 5803-the test is to be conducted in the State Institute for Experimental Therapy (Paul Ehrlich Institute) Frankfurt am Main.

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control it would not be possible to prevent incidents of the sort mentioned, i.e. which are primarily conditioned by the peculiarity of the milieu into which the vaccine is introduced. However, increases in virulence as well as possible bacterial contaminations, are not in themselves hard to recognize, so that by this surveillance a further safeguard for the harmlessness of BCG vaccination is provided. The safety of the procedure should thus, in normal circumstances, be subject to no serious doubt. It will nevertheless be imperative to examine carefully every single candidate for vaccination, as to whether he has recently passed through any infectious sickness caused by virus (cf. Englebreth and co-workers, or Cross), or whether other circumstances are present, owing to which there must be taken into account a lowering of resistance against the attenuated tubercle bacilli employed in the vaccination. Even more significant than the question of safety, which we may consider as settled-as regards the actual hazard-is the problem of the efficacy of BCG. The experimental bases of our opinions on the immunizing efficacy of the vaccine material are not known widely in medical circles. Fundamental weight is given to the investigations of Calmette 1 who, even before the First World War, was able to establish that laboratory animals that are subcutaneously vaccinated with the "gall bacillus" acquire a definite protection against subsequent infection with virulent tubercle bacilli. Calmette's results have been confirmed and established by numerous investigations, in Germany particularly by Neufeld in 1930, but were almost unanimously overrated as to their significance; they ought to be completed by accurate data as to the degree of protection attained. We mention here in particular the works of Seiter and of Seiter and Weiland, who reported a temporally limited, complete immunity in guinea-pigs. And we give as follows the original words in which Bruno Lange 3 (p. 324, loc. cit.), one of the most thorough scholars in the difficult field of experimental tuberculosis research, has expressed the state of affairs; his presentation coincides with our own observations in every respect. Guinea-pigs and rabbits, after parenteral pre-treatment with BCG culture, acquire a heightened power of resistance against a virulent infection. This heightening of resistance appears with special clarity when opposing an infectio minima corresponding to conditions of natural contagion. It manifests itself in a more or less pronounced restriction of the test infection. The pre-treated animals generally live longer than the controls; but nevertheless, they all finally become tuberculous and perish, like the controls, from their tuberculosis.... Only Gerlach and Krause could affirm strikingly favourable findings in

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monkeys; Stanley Griffith, on the contrary, could demonstrate no protection against artificial or natural infection with virulent bacilli in monkeys pretreated with BCG, whether subcutaneously or orally. The investigations of Kalbfleisch and Nohlen, of Tzechnovitzer, and of L. Lange, ran an equally negative course. Other authors had irregular findings of immunization . . . . Investigations in the Robert Koch Institute ... indicate ... that subcutaneous pre-treatment with BCG confers on cattle and sheep a protection against a not too severe test infection, though not at all regularly. Calmette's vaccination was distinctly inferior to Koch's preventive vaccination.

These conclusions have subsequently been sustained time and again by investigations using animal experiments, on which Spiess2 has made a comprehensive report (cf. in particular Helmert; Klose and Dontenwill ; Spiess1 ; et al.). The great significance of bovine tuberculosis in the epidemiology of human tuberculosis appears readily from the fact that about 10 per cent of all forms of the disease are derived from the typus bovinus (Brugger; Freese; Goerttler and Weber). As is well known, it came to the fore in Calmette's first attempts at vaccination. Verification of his favourable results by numerous investigations all over the world (cf. Neumann 1) led, however, to very contradictory, often negative, findings (Ascoli; Dalling; Fliickiger; Kaplan; Meyn; Ramon 1 ; Seelemann and Rackow; Uhlenhuth 1- 3 and co-workers; Zunker). The International Bureau on Animal Diseases in Paris drew conclusions from these facts in 1948. In a statement published at that time, it declared, inter alia: Even if we have succeeded experimentally, by inoculation of tuberculous antigens of whatever sort, in inducing specific antibodies in the animals thus treated, none of the preparations, including BCG, that have so far attained application, has justified itself in practice. Hence, the nations that are most advanced in the theatre of the fight against tuberculosis, have entirely discontinued attempts at preventive vaccination, in so far as they have ever even attributed any importance to efforts of this sort. They lay the main stress on prescribed hygienic measures. In view of the fact that infections launched through natural channels induce no adequate immunity in the animals (these are not protected against a subsequent infection of external or internal origin) it is improbable that we can make use of the same principle to build up an effective method of protection .. (Quoted from Ramon 1, loc. cit., p. 50.)

Thus, vaccination was disposed of for veterinary medicineseemingly for good. Although, in the final analysis, the same restrictions must be laid on the problem of preventing human tuberculosis, in this case it has

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not been possible, for obvious reasons, to decide to give up the method of BCG vaccination. We can estimate from the publications of recent years that more than 100 million children and young people have been vaccinated since the introduction of Calmette's vaccination-and particularly since the World Health Organization has had mass vaccinations of vast proportions carried out throughout the world. Even if no compelling conclusions can be drawn from it as to the efficacy of protective vaccination, this unique material nevertheless yields a series of suggestions and problems for further work in this field. The usual modern technique of human BCG vaccination is that of intracutaneous injection (Wallgren 1); oral administration (feeding) is almost exclusively confined to South America (Corbach and Lubetkin; Rosemberg4), and subcutaneous led to annoying vaccination reactions on which account it has been discarded. Following intracutaneous administration, there develops, in the course of a few weeks, a vaccination papilla the size of a pea at most, to which is attributed the function of a weak primary dermal infection incapable of generalization and characterized by a strong tendency to regression. In the overwhelming majority of vaccinated subjects (about 95 per cent), there develops within about 4-8 weeks or even somewhat later, a positive tuberculin reaction, the duration of which is very diversely estimated and has been reported as from a few months (cf. Heaf2) up to ten years (Aronson 4 ; Baumann2 ; Daelen and Liltgerath). The duration of demonstrable (vaccine) allergy depends only in part on the dose (Palmer2) and preparation of the vaccine employed, or on the persistence of the (vaccination) focus of infection and the reaction potential of the vaccination subject, which appears to decrease markedly with advanced age (Caplin, Silver and Wheeler; Beresford). The technique of tuberculin testing is also of great significance. In particular, the dosage of tuberculin (tuberculin units) employed for the skin reaction can cause extraordinary fluctuations in the outcome of the reaction, whence allergizing effects can be overemphasized or else overlooked that would have been properly demonstrated by an appropriate dosage. Thus, Edwards and Getting found 100 per cent of vaccinated children tuberculin-positive with 100 TU but only 36 per cent with 5 TU. Hence, uncontrolled losses of potency must have grave consequences (Edwards and Dragstedt; Guld, Magnus, and Magnusson; Schmidt-Rohr and Winnewisser). Zonal phenomena (Kaneku) also give rise to faulty interpretations in certain circumstances; and different reaction findings can result from the use of

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different tuberculins (old-tuberculin or GT) (Gotzky; E. Jensen; Kleinschmidt3 ; Kolitz; and others). These discrepancies, which can be further increased by the use of different methods of performing the test (Moro, Mendel-Mantoux et al.), make statistical surveys more difficult both in the pre- and in the post-vaccinal phases. To this is added the danger, increasing in degree with prolonged periods of observation, of having the vaccine allergy overlaid by superinfection with virulent pathogens, which on its part would then lead to a rise in allergy. Thence results a substantial drawback-as regards early diagnosis of tuberculous infection and epidemiological investigation-of BCG vaccination: the alarm signal of incipient tuberculin hyperergy is submerged in the vaccine allergy, especially when this attains a high degree (Gorgenyi-Gottche; Heaf; WHO-TBC Research Office). The possibility of differentiating the BCG and the infection allergies has indeed been asserted (Epstein; Ruziczka) but must be treated on the whole with great scepticism (Lorenz and Quaiser; Stejnberg; Zeh). The employment of BCG tuberculin (Dedebas; Magnusson, Lithander and Hagberg; Quaiser), of fractions thereof (Toda), or of the BCG test that initiates Koch's phenomenon by injection of vaccine (Aronson and McGettigen; d'Avignon; Fourestier and Blaque-Belair2 ; Friedman and Silverman; Ustvedt and Aanonsen) have certainly led to a rise in sensitivity and in specificity of the allergy tests but not, however, to the possibility of distinguishing the different allergies induced by BCG and by virulent pathogens respectively. Thus, by rigorous agreements on the modus procedendi, prevaccinal allergy can probably be analysed statistically with fair exactitude (Freudenberg), whereas the post-vaccinal is subject to a series of influences hard to evaluate. On these grounds, reports on protracted vaccine allergy are to be interpreted with caution, especially since both clinical (Anderson and Palmer; Linneweh and Luther) and experimental (Degli Esposti; Weiland) observations have shown an occasional rapid disappearance of the sensitivity or immunity. Naturally, individual readiness for reaction plays a role that should not be underestimated even here. For wide-ranging surveys, we must also bear in mind-especially when heavy doses of tuberculin are being used-the danger of nonspecific and regionally distinctive consequent reactions (Aronson 3 ; Palmer3 ; Palmer, Krohn, Manos and Edwards). Thus, Palmer describes a striking prevalence of individuals reacting weakly (only to large doses) in certain geographical regions.

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Nevertheless, in all discussions concerning the degree and duration of the tuberculin allergy attained, we must always keep in view that we are here dealing with questions of secondary importance for the goal towards which we are striving. Since hypersensitivity towards tuberculin can qualify only as an expression of hypersensitivity towards one of the antigens formed by the tubercle bacillus, but not as an expression of a universal allergy or immunity, the positive tuberculin reaction offers no testimony as to whether the subject of vaccination has acquired henceforth a defence against tuberculous infection. Thus, matters stand otherwise than under natural conditions, in which infection, immunity, and tuberculin allergy always appear in combination-even if in variable relationship. Since, now, one antigen may suffice to induce tuberculin allergy, the deduction from a positive tuberculin reaction of an immunity acquired under artificial conditions, is not permissible as a matter of course. We are, rather, obliged to draw our conclusions as to the immunizing activity of BCG vaccination from other sources of information, before we can assume that we have the right to see in the positive tuberculin reaction, even simply the "indicator" of a success£ ul protection through vaccination. Here, only a statistical study of vaccination results satisfying all critical demands, can provide a clear picture. Whereas, for example, the results of preventive vaccination for diphtheria have been for years, in Germany and elsewhere, tested in the most painstaking way and, in particular, even analysed by the methods of mathematical statistics (Carlinfanti and Molina; von Schelling; Wohlfeil), the results of tuberculosis vaccination with BCG are today, almost without exception, evaluated according to more or less subjective impressions. We almost entirely lack a truly satisfactory, critically scientific analysis of the material obtained in numerous mass vaccinations; and on the other hand, the material is not infrequently obtained in a way that excludes such an analysis in the first place. The essence of follow-up statistics in BCG vaccination lies in a comparison between the incidence of illness or death from tuberculosis among the vaccinated and the unvaccinated. In making it, the majority of authors have been satisfied to establish a difference between the morbidity and mortality figures in the two groups, but have neglected to ascertain whether these differences were really due to the BCG vaccination or whether other distinctions existed between the compared groups, which would have a bearing on the course of a tuberculous infection. In spite of the findings of numerous authors arguing in favour of it (Aldershoff and Heynsius; Anderson and

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Belfrage; Aronson2 ; Birke; Birkhaug3 ; Ferguson2 ; Hyge 1 ; Klebanow; Marfan; Nordwall; Rosenthal1·2 ; Saye 1 ; Scheel 2 ; Tornell; Turk; Weill-Halle; Weleminsky; et al.), the problem of the effectiveness of BCG vaccination must be considered as still unclarified. On this point, even works appearing in recent years, some of them based on very large research material, have not succeeded in changing anything. If an author recognizes only such investigations "as meet the standards of a laboratory experiment"-as do van Deinse and Griesbach-he could not recognize as valid a very large part of the statistics on the immunizing activity of BCG vaccine. This would be in part because of the smallness of the vaccination figures on which they are based, in part because of a distribution of vaccination subjects which was not according to chance, or because of other mistakes in the experimental set-up influencing the results (Daelen and co-workers; Fourestier; Baisette and Blaque-Belair; Fischer; Gernex-Rieux, Gervois, Ramon and Tacquet; Hadvall; Heimbeck2 , whose conclusions were strongly criticized especially by Wilson; Kallabis; Kereszturi and Park; Konig and Schulze; Kristenson; Olsen; Savilahti; Vojtek; Wallgren 2 ; et al.). Even the well-known investigations of Dahlstrom on several annual classes of recruits are fundamentally questionable because of the voluntary nature of the vaccination and the resulting possibility of selectivity in the contrasted groups; although, in this particular case, the thorough statistical working-out of the large material appears to admit the equivalence of the vaccinated group with the non-vaccinated. Dahlstrom deduced from his survey a certain protection effected by the BCG vaccination against primary tuberculosis, whereas for postprimary forms of the sickness, no statistically significant distinction between the two groups could be made. The five-year hazard of pulmonary tuberculosis stood even higher for the vaccinated than for the controls-from which, of course, one ought not exactly to draw the inverse conclusion that the BCG vaccination predisposes to postprimary infections. One must attribute a certain indicative value-even though limited because of the usually small numbers-to observations on aggregate infections deriving from one source of infection in communities which were only in part vaccinated with BCG (Dickie; Dietzsch; Haefliger2 ; Henkel; Holm 2 ; Hyge2 ; Pampe) . Among the most frequently quoted BCG statistics of recent years, belong the investigations of Aronson and Palmer, of Aronson and Ferguson-Aronson1.2, and of Stein and Aronson, on North American

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Indians. These were soundly planned and statistically impeccable works, in the course of which altogether about 1500 young tuberculinnegative subjects in the age-group of 1-20 years were, in alternate cases, vaccinated with BCG and sodium-chloride solution, and observed for fifteen years; they seem to show that with a consistently equal mortality from non-tuberculous diseases, the tuberculosis mortality of those vaccinated was reduced to about a sixth. Further observations on the Indian children vaccinated with BCG at that time also seem to confirm the preliminary results (Aronson 4 ). Certainly the immunity falls off markedly, ten to eleven years after the vaccination; this is recognizable by the cumulative death-rate of the vaccinated group, which then sharply increases. Wilson adduced against Aronson's deduction that the Indians have a genetic, constitutionally lower power of resistance towards tuberculosis, and thus a higher hazard of infection on exposure, than do whites, so that the general validity of the conclusions drawn was questionable. In spite of the favourable outcome of the "Indian statistics," Palmer 1, for many years a co-worker of Aronson's, expresses himself very sceptically in his report to the World Health Organization regarding methods of investigation in mass BCG vaccinations: " ... it must be stated in clear and unequivocal language that we have not at present a truly scientific proof for the efficacy of BCG as a preventive against human tuberculosis. This agrees with the facts, although in the past 20 years millions of human beings have received BCG and in spite of the very impressive findings of a few isolated investigations carried out on a small scale .... " The investigations carried out by Palmer and Shaw in four experimental series on nearly 300,000 individuals gave as a result that a great number of the cases of tuberculosis occurring in the first years after the vaccination programme, fall in the group of unvaccinated persons who had been previously infected through natural channels. Between those vaccinated and those not vaccinated no difference was to be demonstrated either in tuberculous morbidity or in mortality. Of course, the total number of sicknesses is so small that, in the view of the authors, the question of the effectiveness of BCG preventive vaccination is obviously unlikely to be solved in this way. In conclusion, the authors support the opinion shared by most American tuberculosis experts today (cf. also Myers) that there are no grounds to suppose tuberculosis might be more effectively conquered in that country by the introduction of mass vaccination than by the

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otherwise usual prophylactic measures. The American College of Chest Physicians (cited from Young) also maintains the same point of view. In a survey on "The Present Status of Vaccination in Tuberculosis Control Programs," it declares, inter alia: " ... It must be stated that there is no evidence that meets strict scientific requirements demonstrating that BCG effects the controls of tuberculosis, despite the suggestive results of a few studies .... " In this connection, attention must be called to the genuinely "dramatic" regression particularly in juvenile, but also in general, tuberculosis mortality in all civilized nations (de Haas; Hoogendoorn; Kreutzer; McDonald and Springett; Medlar; cf. also Uhlenhuth 4), which cannot be correlated with vaccination against tuberculosis (Abreu; Anderson and Palmer). Furthermore, pathogenesis and epidemiology of tuberculosis indicate that the chief problem in combating it today concerns the tuberculosis of adults beyond the fortieth year of life. The statistics reported by Medlar for the State of New York reveal a regression of tuberculosis mortality from 1905 to 1945 of about 91 per cent in the age group under 15 years, about 77 per cent between 15 and 40 years, in contrast to only about 28 per cent in those over 40 years. According to the statistics for the United States (1947), 26 per cent of all fatal cases of tuberculosis occurred in persons between 15 and 35 years, but, in comparison, 70 per cent in the 40-year age-group, the latter being actually for the most part the result of a reinfection after healing of primary infection. As regards the efficacy of BCG vaccination, it must accordingly seem doubtful whether an artificial immunization with attenuated pathogens should lead us to expect results just where a natural (virulent) vaccination so often fails. If we consider further, in addition to the adverse opinions regarding the efficacy of BCG vaccination, the vaccination risk (cf. Wallgren 8), we can understand the report of Mostyn, recently published in the organ of the National Association for the Prevention of Tuberculosis, regarding the BCG situation in Switzerland and in the Scandinavian countries. This remarks, inter alia: 0

The whole position of BCG vaccination in Scandinavia is being reviewed. Now that tuberculosis morbidity and mortality is so low, many feel that mass vaccination is no longer necessary and that testing for tuberculin conversion is the best means of case finding. In Scandinavia, where morbidity and mortality are low, case finding reads like a detective story. In these countries children are tuberculin tested yearly or biyearly and when any child undergoes tuberculin conversion all his contacts

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are examined for the source of infection. Dr. Gedde Dahl of Oslo stressed what most doctors there feel-that the fall in morbidity is due more to mass X-ray surveys, to early case finding, and the removal of infectious cases, than to BCG, but in countries where it is difficult or impossible to treat all infectious cases and where living standards are low, BCG can play a most important part.

Taking strict account of biostatistical principles, very few publications on the results of BCG vaccination remain that do justice to all requirements for an unbiased treatment of the subject in its entirety; of these we should primarily mention here the works of DeutschLederer, of Epstein!, of Freudenberg, of Greenwood, of Lange3, of Lenz, of Levine and Sackett, and of Rosenfeld. Especially typical seem the investigations of Levine and Sackett, who have not only treated the topic in outstandingly critical fashion, but have also fulfilled conditions which alone, generally speaking, make possible an unimpeachable working-out of the problem. The investigations carried out under the authority of the Department of Public Health of New York City come under two main headings. During the first period reported (1927-32), the children considered for vaccination were divided into two groups : the children of one group were vaccinated with BCG, those of the second (control) groupwhich otherwise received equal care-were simply kept under observation as to their state of health. Now whereas, of the 445 vaccinated children, only 3 (0.068 per cent) died of tuberculosis, of the 545 children in the control group, 18 (3.38 per cent) succumbed to the results of tuberculous infection. The tuberculosis mortality among the unvaccinated was thus five times as great as among the vaccinated! The difference between the two groups amounted to 2.70±0.89, and was thus established as statistically significant (T = 3.04 according to H. von Schelling). Nothing could be more plausible than the assumption that the source of this difference in the behaviour of the two groups was the vaccination . And yet, it was demonstrable that there had been a false conclusion and that along with the "vaccination" factor was linked another factor which had vitiated the apparent result in advance. The investigators finally came to the conclusion that, on grounds of previously unnoticed circumstances-e.g., parental co-operation, social status, influences related to race, etc.-an unintentional preferential selection was being made of such children as had grown up under conditions more favourable for health; they then, in the second period reported (1933-43), changed the manner of group formation, so that an unbiased selection of vaccination subjects was absolutely

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guaranteed: the selection was left entirely to chance, by actually vaccinating, in strict alternation, only every other child registered and considered for the vaccination, so that any discriminatory manipulation of the two groups was excluded. Subsequently, the second section of the vaccinations led to entirely different results: of 566 vaccinated children 8 ( = 1.41 per cent) died of tuberculosis, of 528 not vaccinated, likewise 8 (1.51 per cent). The difference between these two groups amounted to 0.10±0.70 per cent, and thus fell well within the limits of chance variation for this sort of observational series. Serious arguments cannot be brought against these results. Holm 1 did indeed raise the objection that, in Levine's statistics, a case of fatally terminating broncho-pneumonia following whooping-cough had been counted as a death from tuberculosis following vaccination; and he expressed the opinion that the acid-fast bacteria found in the mediastinal glands were nothing else than BCG bacilli escaped from the point of vaccination. Nevertheless, Levine's work established explicitly that the bacilli discovered belonged to the human type, whereas BCG is well known to be of bovine origin. Furthermore, the case in question belonged to the first period reported in which, as a matter of actual fact, fewer cases of death from tuberculosis were demonstrable in the "vaccinated group" than among those not vaccinated. Hence, nothing is changed in Levine and Sackett's results, even if we assume that of the 445 vaccinated cases, not 3 but only 2 (0.45 per cent) died of tuberculosis. Of very much greater weight is the consideration advanced by Kleinschmidt1 and by Kuster in opposition to Levine, that the New York children had continued to remain in their usual environment after the vaccination, and thus had already been exposed to infection before development of the vaccine protection. But this objection too cannot be sustained; for it assumes that danger of infection existed only in the period immediately after the vaccination. However, since the incidences of onset of infection were distributed over the whole duration of the observations, it should nevertheless have been possible to establish an absolute difference between the tuberculosis mortality of the vaccinated and unvaccinated groups, if the vaccination had effected a noteworthy protection. Omission of isolation in the critical period could have effected only a decrease of this difference but in no case have made it disappear. Furthermore, Levine and Sackett have also carried out a series of vaccinations in which the children were kept in a tuberculosis-free

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environment for three months before and after the vaccination. Here, of 91 vaccinated, 1 (1.1 per cent), of 96 not vaccinated, 3 (3.1 per cent) died; the difference amounted to 2.0 ± 1.98 per cent, and thus fell entirely with the limits of chance (T = 1.01) . This series was too small to provide a real clarification. The proper criterion of Levine's investigations is to be seen in the similarity of the groups to be compared. If one leaves the unvaccinated group of control individuals in their habitual environment, then a possibility of comparison exists only if those vaccinated were likewise not isolated . If, on the contrary, those vaccinated were isolated-which every practitioner will very rightly require-then the control individuals must also be isolated in the same way and during the same length of time. Thus, the procedure of alternation employed by Levine and Sackett was, on the whole, not affected by the question "to isolate or not to isolate." Martini has rightly emphasized this prerequisite in his Methodenlehre der Therapeutisch-Klinischen Forschung. In recent years, the report of the Medical Research Council of Great Britain, "BCG and Vole Bacillus Vaccine in Prevention of Tuberculosis in Adolescents," has aroused great interest since-when comparing them to alternative procedures in an unimpeachable statistical manner-it appears to give an unequivocal indication of the preventive activity of BCG (and of the Vole bacillus). The tuberculosis morbidity of 13,300 tuberculin-negative 14½ to 15-year-old adolescents, was compared with the annual morbidity rate of 14,100 initially tuberculin-negative subjects vaccinated with BCG, and 6700 vaccinated with Vole. The evaluation by X-ray control gave, in comparison to the unvaccinated tuberculin-negative cases with an annual morbidity rate of 1.94 per thousand, 0.37 per thousand for those vaccinated with BCG and 0.44 per thousand for those vaccinated with Vole.* However, weighty objections were raised by Myers against the evaluation of this experiment. Since there is a lack of differentiation between spontaneously relapsing primary complexes, and post-primary manifestations, we can no longer evaluate experimentally the protective action of BCG. On a comprehensive presentation of the clinical picture of the primary lesions, Myers bases his criticism, which culminates in the following summary: Apparently it has been the failure to properly classify pulmonary lesions that *In addition, 8200 tuberculin-positive adolescents were observed. In this group, 1.75 per thousand of those individuals reacting positively to 3 TU, sickened annually, 0.74 per thousand of those subjects responding only to 100 TU.

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has led to the remarkably uniform statement in many reports in the literature to the effect that four or five times more controls develop lesions than those who have received BCG. The number of primary infiltrates that attain shadowcasting proportions soon after natural initial invasions occur is enough to account for the greater part of this difference. This is good reason to believe that enough primary infiltrates were reported as "definite cases of tuberculosis" by the Clinical Trials Committee to account for most of the difference between the controls and those who received BCG or Vole bacillus. One would expect more of those who acquired natural infections to present primary infiltrates in the lungs than those who received BCG or Vole bacillus and thus had primary complexes in the skin and the regional lymph nodes . . ..

Myers thus denies that the number of true tuberculous lung infections is substantially higher among those not vaccinated than among those vaccinated. He maintains that the shadows observed are for the most part only shadow-casting primary infiltrates. That the number of shadow-casting primary conditions is substantially greater in the non-vaccinated than the vaccinated subjects is referable, according to Myers, to the fact that the latter have their primary involvements in the skin and the regional lymph nodes. By this, of course, Myers admits implicitly that non-virulent BCG is capable of preventing or "displacing" the development of virulent tuberculosis primary complexes in the lungs. It mav well be also that those individuals whose immunization is referable to specific pulmonary primary infiltrates, and who already harbour virulent bacilli in their systems, are actually in a somewhat less favourable position than the vaccinated subjects who are threatened only by exogenous infection. Yet these distinctions hardly seem to carry much weight. Myers considers that even the virulent primary infiltrates are generally of little significance, and regards only the post-primary changes as substantial sources of clinical developments. From the works discussed, we ought not to decide, without further confirmation, that BCG vaccination should be judged worthless. The one really warranted conclusion from the numerous investigations undertaken up till now appears rather to be that the value or worthlessness of the method is still undecided. The same conclusion was recently drawn by Grigg. She probably also reflects the opinion prevailing in the United States (Palmer, Shaw and Comstock) which is also clearly expressed in a publication issued in 1958 by the Trudeau Society on "The Role of BCG in the Prevention of Clinical Tuberculosis." In no case is the protective value of BCG vaccination to be reckoned as very high. This is especially so since BCG, which belongs to the

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typus bovinus-in spite of its practically undiminished ability to form tuberculin-produces no phthionic acid (cf. chapter IV), and so does not possess an antigenic element that is especially characteristic for human tubercle bacilli in particular (Roulet and Brenner); and it is supposed to produce even similar compounds only on a small range-in accordance with its low virulence. In the most favourable case, we should be able to assume that the artificial infection established by BCG vaccination is capable of substituting for a primary infection, and producing just such an immunizing effect as would the latter. With individuals vaccinated with BCG, about 5 per cent of the cases do not even acquire a positive tuberculin reaction, whereas a true primary infection produces a tuberculin allergy almost without exception; hence, we must assume that the protective effect to be expected from BCG will be less frequent, weaker, and of shorter duration than the defence that arises from the resolution of a natural infection. Medlar et al. have also pointed this out in various ways. We should, therefore, expect no more than that the manifestations of an active primary stage would be spared to a greater or lesser portion of vaccinated subjects who are exposed to a superinfection with virulent bacteria, and that they will get through the course of the superinfection without objective and subjective symptoms. This effect would be one to regard as even very significant; but it remains to be seen whether vaccination projects, on the pattern of the procedure inaugurated by Levine and Sackett, will be carried out in future so that an objective decision will at last be possible as to whether this effect can be attained. We have already pointed out that the immunity of the tuberculous organism does not suffice to kill off tubercle bacilli penetrating afresh from without, or to rob them of their virulence; they are only prevented from multiplying. Bacteria invading after a BCG vaccination thus retain their dangerous character, and behave nearly as do the bacteria remaining in the body after a primary infection has been overcome: i.e., they represent a life-long threat to the host organism, which can become acute upon any sort of fluctuation in the powers of resistance. Thus, the vaccination can provide no protection against the secondary and tertiary manifestations of tuberculosis as, indeed, the extensive investigations of Dahlstrom have shown (cf. also Griesbach; Haefliger3). The course run by the disease after the establishment of the superinfection with virulent tubercle bacilli, is rather the same as after the healing of a natural primary infection and depends on the

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interaction of the non-specific powers of defence (Prigge 2) with the specific powers acquired as a result of the reaction of the macroorganism against the pathogen, as expressed in the "resistance" of the organism (Kolle and Prigge). We should probably also take into account that the post-primary hematogenous manifestations (the appearance of which seems to depend, up to a certain point, on the extent of the primary complex), may well occur more rarely in those individuals successfully vaccinated than in those not vaccinated; this is regarded as particularly significant for tuberculous meningitis (Catel and Daelen2). Nevertheless, even Wallgren 9 takes a very cautious stand on this question: "Probably BCG vaccination also to some extent reduces the risk of early post-primary tuberculous manifestations, in particular meningitis and miliary tuberculosis." Likewise, Daelen's2 statistics on the "Morbidity and lethality of tuberculous meningitis in the years 1947-1951 in Hesse," frequently cited in Germany in recent years, allow no sure conclusions as to the effectiveness of BCG vaccination on grounds of the material employed. The groups compared have not been selected on a chance basis, nor does the evaluation take into account the epidemiology of tuberculous meningitis; the numbers of meningitis cases in 3-18 year-old subjects were compared for those vaccinated and those not vaccinated. Since, however, in the vaccinated group, the number of children under 6 years is exceedingly small, and yet the age-peak of tuberculous meningitis lies in the third year of life, it is no wonder that fewer cases occur among those vaccinated than among those not vaccinated (Lydtin, unpublished). But not only faulty planning of an investigation or evaluation of results can lead to false conclusions; systematic influences such as unequal distribution of resistance in two groups to be compared (LydtinL 2 ; Freerksen and Rosenfeld) or epidemiological developments can also lead to false conclusions. "The fall in the rate of primary infection in childhood has led to a decrease in the incidence of childhood tuberculosis. Since meningitis and miliary tuberculosis mainly affect the youngest children who nowadays rarely become infected, the decrease in these two generalized forms of tuberculosis may be explained solely by an elimination of the risk of exposure; BCG vaccination may not necessarily have anything to do with the disappearance of these diseases .... " (Wallgren9). A similar statement is made by Gedde-Dahl, who replied verbally to the contention of several authors that the regression of tuberculosis morbidity after the war was a result of BCG vaccination: "There is

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no statistical or epidemiological basis for this"; and he furthermore substantiated this point of view. We must consider as hypothetical that protection can be extended to the later stages of tuberculosis in addition to the early post-primary manifestations of the disease, as Wallgren 3 and Baumann 1 believe; and clinical experiences seem rather to speak against it (Simon Jr.). As a prerequisite for the statistical evaluation of BCG vaccination we must require that the vaccination candidate should tolerate, without reaction the intracutaneous injection of 100 tuberculin units (Tu) and, so, be "tuberculin-negative." The recently proposed limitation to 30 Tu must rate as questionable since, with this technique, tuberculous subjects with a weak allergy might possibly be passed over, yet might develop a considerable immunity which, since it is not due to vaccination, would necessarily lead to falsification of the statistics. The general question whether the vaccination is permissible for already tuberculous subjects, need not be gone into here. It is held to be harmless by Latin-American advocates of the undoubtedly less radical oral method of administration (Assis; Balteanu and Toma; Fernandez and Sivila; Loleo; Silveira and Medeiros; all cited from Rosemberg2), and also by Wallgren 6, by Cervia-Cabrera, by Fitzpatrick, by Gernez-Rieux, Piat and Voisin, and by Quaiser; whereas Destexhe, Kostic-Yoksic, and Levi-Valensi and Migueres, among others, are expressly opposed to the vaccination of persons already tuberculin-allergic, because of the danger of activating existing foci by the BCG superinfection (Koch's phenomenon in the form of the focal reaction). This view is subscribed to by the majority of those investigators who use parenteral BCG vaccination, and finds expression in the requirement mentioned above, that the vaccination candidate demonstrate the absence of a tuberculous infection by his tolerance for even high doses of tuberculin (cf. also Kleinschmidt4). Since the tuberculin reaction is still negative in the first weeks following an infection ("pre-allergic" phase), a negative result certainly lacks full indicative value if the children live in surroundings with a tuberculosis hazard; so that it would be desirable to isolate the tuberculin-negative children who are under consideration for BCG vaccination in tuberculosis-free surroundings for some time (up to three months) and to repeat the tuberculin test before carrying out the vaccination. As a control of the results, the tuberculin-negative subjects vaccinated with BCG are subjected to a second tuberculin test after eight to twelve weeks and, in case this is not positive, vaccinated again

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with an increased dose. If at all possible, the isolation of the children should be continued until the positive tuberculin reaction appears. In those countries in which the vaccination candidates are selected on the basis of their negative tuberculin reaction, the appearance of the post-vaccinal tuberculin allergy is generally considered as an indication of the establishment of an immunity (Frappier, Guy and Desjardins; Heafl; Heimbeck2 ; Monaldi 2 ; Wallgren 7 ; Yanagisawa; cf. also Palmer and Edwards). Since, however, the allergy persists, on the average, only four yearsif no virulent superinfection ensues-Wallgren recommends repetition of the tuberculin test at the ages of 3, 7, 10, 15, and 20 years, i.e., at the ages particularly susceptible to a tuberculosis infection, and renewed BCG vaccination when indicated. However, we must clearly understand that even the most careful adherence to all these precautions can, by itself, give no information as to what the vaccination procedure has achieved. We may expect clear indications of this only from an alternate vaccination carried out under strictly controllable conditions. (c) Vole bacillus If observations made in different ways verify a progressive attenuation of the virulence and, thereby, of the antigenic efficacy of BCG, we will necessarily have to look about us for other immunizing strains or for other procedures. From this point of view, the Vole bacillus discovered in 1937 by Wells deserves special attention. This bacterium, highly virulent for Vole mice, and identified by Brook and Wells2 •4 as a tubercle bacillus belonging to the typus murinus, certainly shows certain morphological and cultural, but no serological differences from the classic type (Wells and Wylie2). Also, the Vole bacillus tuberculin is qualitatively indistinguishable from the human. The pathogenicity of this pathogen is only slight for the usual laboratory animals, and it requires excessive subcutaneous doses to induce progressive tuberculosis in guinea-pigs, this being then actually indistinguishable from the tuberculous infections invoked by human or bovine germs. In rabbits, relations follow a similar pattern (Brooke; Griffith; Wells2 ; Young and Paterson). The tuberculin allergy produced by Vole vaccination is said to be pronounced and persistent and to exceed that attainable by BCG. Hall and Wylie, using the technique of multiple scarification, which rarely leads to local complications, were able to attain 100 per cent

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conversion of the tuberculin reaction in small children by means of the Vole vaccine; in school children and adults-tested with 10 Tu tuberculin-they attained 70 per cent, compared to 40 per cent after BCG, although the latter was administered in a concentration (20mg/ccm) ten times that of the Vole vaccine (2mg/ccm) (cf. Wells and Wylie 2). The work of Murohashi, Seki and Tekano, and of Sula and Kombercova also demonstrates a higher antigenicity-as measured by tuberculin allergy-for the Vole bacillus as compared to BCG. The interrelations between allergy and immunity will not be discussed here a second time. Wells and Wylie2 maintain the point of view, already discussed in the immuno-biological chapter, that the two phenomena are not linked, since they observed guinea-pigs that became tuberculin-negative again after Vole immunization, yet retained, over a month, the same power of resistance against virulent infections. The local reactions following Vole vaccination are severe with subcutaneous administrations, yet are said to be unusually rare in intracutaneous injections or multiple scarifications. Generalizations have not yet been observed in man-even in thousandfold overdosages (Sula, Zavadilova, Medulanova and Pokorny; Wells and Wylie2). Sula and Kombercova expressly refer to the good compatibility of the Vole vaccine, whereas M9lller observed more severe reactions with it than with BCG. In view of the apparently higher antigenicity, or virulence, of the Vole bacillus, we should probably expect more frequent and severe local reactions, even from the theoretical standpoint; likewise, we should assume that the immunizing index (so to call the interval available between non-infective immunizing effectiveness and pathogenicity) with Vole vaccine, will be smaller than with BCG. The fact that no generalized infections-as with BCG-have been observed, can be referred to the small extent of the statistical material available. On the basis of experimental foundations and theoretical reflections, we may assume that the degree of immunity against virulent infections to be attained with the Vole bacillus is about equivalent to the protection attainable up to now with BCG (Birkhaug2). However, the statistical foundations are too slight to enable us to make even approximately accurate statements about them. Even the report of Wells and Wylie2 on 2500 Vole-vaccinated subjects with a tuberculosis morbidity of 0.085 per cent, and 700 "comparable" controls with a morbidity of 2.35 per cent, does not alter the fact that, so far, no assured declarations can be made either as to the absolute immunizing powers of the Vole vaccine or as to Vole/BCG comparisons.

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Above all, there is no question of wishing to demonstrate anything like a superiority of the Vole vaccine over BCG. It appears, however, to be of great importance that we possess, in the Vole bacillus, a bacterial reservoir to which we can have recourse in all respects, were BCG really to fall off in its immunological capabilities; in which case, the fact that the Vole bacillus need not be attenuated by difficult manipulations and held at a prescribed level of virulence (the maintenance of which can never be guaranteed with BCG, cf. van Deinse3) is a distinct advantage. The strain discovered in 1937, which has since been continuously passed down through the host animal, has today still the same virulence as when isolated (Wells3). Hence, prosecution of the work on the Vole bacillus must be regarded as an important duty of tuberculosis research, as long as we have at our disposal no other procedure for immunization than BCG vaccination or other methods working with attenuated, living pathogens. 3. VACCINATION WITH KILLED TUBERCLE BACILLI Robert Koch 2 himself undertook the attempt to immunize guineapigs against virulent pathogens, by treatment with killed, dried, and pulverized tubercle bacilli, and was not wholly unsuccessful in this. Koch started from the premise that the germs used in the vaccination could be effective only if, as in the case of miliary tuberculosis, they penetrate to as many organs as possible and act on them; hence, his effort was directed towards heightening the resorbability of the bacteria by pre-treatment with acids or alkalis. Other authors saw an essential condition for the establishment of the immunizing effect, in the use of highly conservative procedure for killing; for example, Lowenstein 2 seems to have achieved certain results from vaccination with cultures that had been killed by the action of light continued over a year, and so did Di Donna. Later, Lowenstein, using formol-bacteria, and Branch and Enders, using heat-killed germs, obtained similar results. The investigations undertaken by numerous other authors, which now have only historical interest, need not be gone into here. Fundamental significance should be attributed to the discovery made by Romer2 that guinea-pigs pre-treated with large quantities of killed tubercle bacilli, become tuberculin-sensitive (cf. chapter III) . This finding has been confirmed by various authors, particularly by Bessau 2 ; and Bessau, as well as Langer, and Opie, Flahiff and Smith, have also succeeded in inducing a positive tuberculin reaction in man, with killed bacteria. Even isolated cell walls of tubercle bacilli appear

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to possess allergenic properties (Ribi, Larson, List and Wicht). The discussion, arising as a result of such investigations, as to whether the tuberculin allergy and rise in resistance following administration of killed pathogens is "the expression of a specific immunity or a nonspecific activation" (Uhlenhuth2 ) could not be decided with certainty from the diverse findings (Adam; Bessau 1 ; Mastbaum; Meyer; Uhlenhuth and Jotten; and Yu). Nevertheless, the specific component undoubtedly plays a leading role in the anti-tuberculosis reaction, even if it is to some degree masked by non-specific factors. The investigations of Bessau 3, from whose viewpoint the dead bacteria precipitate a "slight tuberculous primary complex," also show that animals rendered allergic have a certain protection against virulent bacteria. Lange, Freund and Jochimsen, and Zinsser, Ward and Jennings came to similar conclusions. They showed that guinea-pigs and rabbits could be protected by heat-killed germs against a light infection with virulent bacteria, and that this immunity is not fundamentally different from the condition induced by living germs, but is only weaker and of shorter duration. Similar findings were also reported recently by Helmert, who attained the same protective effect with heat-killed germs as with BCG. The experiments of Coulaud 2 , Haefliger 1, HenseJI, Lagrange1, Straus, and Saenz and Canetti1 have made possible a further step forward. They have led to the recognition, important even in theory, that the immunological efficacy of killed tubercle bacilli can be substantially augmented by non-specific activators. Even fifteen years ago, Straus was able to show that the protective power of diphtheria vaccine material, in particular of the so-called formol-toxoid, is greatly heightened by adding a mixture of lanolin and olive oil. The same holds true, as has now been shown, for tuberculosis vaccine materials consisting of killed bacteria. In place of the lanolin-olive-oil mixture, used especially by Hensel 4 •5 , which is apparently well tolerated, vaseline or paraffin can also be employed; however, with these, there can arise severe vaccination traumas of local or generalized nature and, in fact, even miliary dissemination and progressive, fatal involvement (Maccone; Rist; Sticotti; cf. also Hensel5) . The hypersensitizing but not pathogenic lanolin-olive-oil vaccine is the basis for an interesting therapeutic suggestion by Hensel 6, who seeks to establish artificial foci in tuberculous patients through subcutaneous vaccination with killed tubercle bacilli, and to incite these to produce antibodies by supplementary doses of tuberculin. Of course, no clinical experiments as to the danger involved seem to be available. Both

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Selter's school (Weiland) as well as Rolle, Kalich and Mayr, and also Wallgren 4 reject the lanolin-olive-oil preparation of killed-vaccine material-though on different grounds. The degree of immunity that the fat-embedded tubercle bacilli induce, seems hardly distinguishable from the protection attainable with attenuated living bacteria in animal experiments; hence, we should carefully determine whether the use of living pathogens (BCG) in man is still necessary and defensible. In very recent times a series of publications, especially Japanese, have appeared on the possibility of employing killed-vaccine material (Ito; Kuwabawa and Yajima; Yamada), also in part on BCG killedvaccine material which, with paraffin or peanut-oil activation, showed the same antigenic or immunizing capabilities as living BCG (Kanai, Hashimoto and Yanagisawa; Yanagisawa, Kanai, Shiga and Ito). In experimental murine tuberculosis, Dubos, Schaefer and Pierce, and Solotorovsky, Gregory and Stoerk, and Swedberg, and Weiss and Dubos1 achieved distinct protective activity with phenol-killed tubercle bacilli, and Hand and Jespersen with urea-killed germs which, however, Aufdermaur and Brodhage could not confirm for guinea-pigs. Comparisons between a formol-killed vaccine and BCG, recently made by Ramon, Dehn~, Richou, Costil, Thierry and Gerbeaux, also showed that activated killed-vaccine material (here using aluminum hydroxide) could bring about results corresponding to those from BCG. Of course, the activator seems able, under certain conditions, to enhance not only the allergizing activity but also the toxicity of the preparation (Weiss and Dubos1). Recently, hyaluronidase in combination with killed-vaccine material has also been employed to heighten the immunizing effect (Degli Esposti1 •4), in part with outstanding results as regards conversion of the tuberculin test. Likewise Salvioli2 reports that, after vaccination with a human-bovine mixed vaccine killed by heat and fortified and lyophilized with hyaluronidase, 92 per cent of the children reacted as tuberculin-positive 6 months after the vaccination, 95 per cent, 12 months after. The results of animal experiments corresponded roughly with those obtained with BCG. On the contrary, results with vaccine material treated and killed with ultra-violet irradiation were significant but mostly evaluated sceptically (Birkhaug and Darvicarrere; Sadelkow; Seagle, Karlson and Feldman; Sarber, Nungester and Stimpert). Among the vaccine materials prepared from killed tubercle bacilli, a special place is taken by Preparation AO presented by Arima,

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Aoyama and Ohnawa. The ideas of these Japanese authors coincide with those of Seibert2 •3 •5 in so far as they attribute special immunizing activity to the proteins derived from the tubercle bacillus (cf. chapter IV) . But whereas Seibert sees in TMS precisely the decisive antigen for inducing tuberculin hypersensitivity, in the view of the Japanese authors the administration of the protein formed by the tubercle bacillus in no way leads to a sensitization against tuberculin. In this connection, we should bear in mind that hyperreactivity of the skin induced by parenteral administration of proteins is not also further expressed by the occurrence, after intracutaneous reinjection of the protein in question, of a reaction corresponding to the "tuberculin" type; rather reactions of the "urticaria!" type occur. On the other hand we have seen that to assume we can induce the same reactions in the skin with tuberculin in animals sensitized with TMS, as in tuberculous animals, appears highly problematical according to Raffel's investigations. Raffel's2 research animals sensitized with TMS were as much as totally refractory towards tuberculin and showed only phenomena of the urticaria! type after intracutaneous injection of TMS. The question has still not been settled as to whether in the course of tuberculous infection there arises in general any noteworthy hyperergy towards TMS, linked with the passage of true antibodies into the blood stream, and whether the true tuberculin reaction of the tuberculous subject is not perhaps triggered only by certain ancillary substances accompanying TMS and its breakdown products contained in old-tuberculin, in purified tuberculin, and in other tuberculins. We may even have to ponder whether tuberculosis immunity may not be so often inconsequential for the very reason that the hypersensitivity developing towards proteins in the course of tuberculosis is too slightin contrast to the hypersensitivity towards the toxic proteins of diphtheria bacilli so decisive for diphtheria immunity.The results obtained with TMS in the United States also seem to require more careful control tests. In view of the criticism of Seibert's tuberculin theory, particular significance should, in any case, be attributed to the results attainable with AO. Of course, the tubercle bacillus does build a multiplicity of proteins as, indeed, the results obtained by SchmidtSchleicher show. It must therefore remain undecided whether, and how far, the proteins contained in the Japanese vaccine material should be identified with TMS. The Japanese authors grow human tubercle bacilli on mediums containing additions of saponins from the fruit-pulp of the soap-nutSapindus mukorji japonica-native to Japan. The culture bacteria

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thus obtained are no longer acid-fast and are said to no longer manufacture any wax fraction. In view of the findings of Raffel2, mentioned above, who claims the wax fraction to be the carrier of the antigenic activity decisive for development of tuberculous allergy, AO vaccine material should claim particular attention. The tubercle bacilli deprived of their wax fraction are kept for two years at temperatures in the neighbourhood of zero, before the preparation of the vaccine material, and gradually die during this time. The laboratory investigations now available on the protective value of the AO vaccine material do not suffice for an objective evaluation; also the human results claimed to have been achieved in Japan with mass vaccinations are in the first place difficult to verify. However, the procedure employed in preparation of the vaccine material, is so interesting that its thorough testing in animal experiments should be rated as urgent. Particular attention should also be directed to the suggestion made by Lembach for increasing the reactions of BCG vaccination in man by a subsequent vaccination with AO. Weiland's objection expressed to this and based on the diverse nature of the principles underlying the methods of preparation of the two vaccines, does not seem to us convincing, since the attainment of a "full" immunity becomes the more probable, the greater the number of antigens against which the defence mechanisms of the macro-organism are directed. Yet all such considerations must be taken only as hypothetical. Evidently, the investigations begun by Arima and co-workers, have not been continued on a larger scale, not even in Japan where the World Health Organization carried out mass vaccinations with BCG after the Second World War. Another killed vaccine, the application of which has been almost exclusively confined to Italy, the so-called Anatuberculina Integrate Petragnani (AIP; Petragnani), has, on the contrary, been able to prove itself even in practice, even though its application has never attained the extent of BCG vaccination. In this connection, we are dealing with a vaccine material variously modified in the course of the years, and prepared from tubercle bacilli of various types, finely ground and killed by treatment with formalin. AIP has been extensively publicized and its use recommended, especially by Mazetti 2 and Mazetti and Kircheis, by Salvioli1, Salvioli and Degli Esposti, and by Signorini and Panero. Investigations making a comparison with BCG (Degli Esposti1; Giovanardi; Mazetti1; Poggi) report an allergizing activity of AIP

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in general somewhat weaker compared to that of BCG; this, after all, we should be able to equalize by means of higher dosage, multipuncture technique, multiple injections, or hyaluronidase fortification. With this vaccine, too, the degree and duration of the tuberculin allergy is largely dependent on the persistence of the "vaccination focus" (Kircheis). For statistical evaluation, the material is neither extensive nor dependable enough. Degli Esposti 1 raises against AIP the objections that are made against killed vaccine material in general: that the development of an effective immunity is dependent on the presence of living pathogens. Of course the justice of this objection cannot thus far be regarded as demonstrated, even though the experiments made with killed BCG (Palmer and Edwards) seem to speak in its favour. 4. VACCINATION WITH BODY COMPONENTS OF METABOLIC PRODUCTS OF TUBERCLE BACILLI

Only a few years ago, general opinion ran that the results of an artificial immunization against tuberculosis could hardly ever equal, not to say surpass, those achieved by nature with the aid of an infection; whereas, the thoroughgoing studies on the chemical and antigenic structure of the tubercle bacilli that have been pressed forward in several countries with great success (bibliography in Prigge 8), permit a certain optimism to arise again, even though the laboratory work is still a long way from findings realizable in practice or, indeed, even from theoretically satisfactory results. The very knowledge that the doctor today can immunize against a series of other infectious diseases better than nature, for example against tetanus, may incline the experimenter to venture to come to grips today with the problem of tuberculosis immunization with cell-free antigens. In particular, we may well hope to know, within foreseeable time, something concrete concerning the antigens decisive for the development of tuberculosis immunity, and to be able to develop methods for their concentration. Even decades ago, great significance pertained to the attempts to utilize, in the immunization of laboratory animals, body substances of tubercle bacilli brought into solution by the aid of numerous compounds-lecithin, neurin, choline (Deycke and Much1. 2 ; Much1. 2 ; Much and Leschke); a preparation produced with neurin is said to have had particular efficacy. Deycke and Much have further attempted to enhance the immune reaction of tuberculous patients so that it

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would become effective even against the bacilli already present in the organism. To attain this therapeutic goal, they subjected tubercle bacilli to prolonged action of lactic acid and obtained, on the one hand, a filtrate containing albumose (MTLB) and, on the other, a protein, a fatty-acid lipoid, and a neutral fatty fraction-"A," "F," and "N,"-by fractionation treatment of the residue left by the cleavage (MTBR). These fractions, designated as "partial antigens," have not indeed fulfilled the expectations based on them; nevertheless, the idea underlying their preparation still enjoys a heuristic significance and now takes on somewhat more precise forms based on our modern knowledge concerning the chemical structure of the tubercle bacillus. Of course, the majority of the investigations presented up to now on the antigenic properties of the chemical building-blocks of the tubercle bacillus are predominantly concerned with the question of how far the immuno-biological reactions initiated by them can be used for their serological characterization and identification (Boyden; Kall6s and Kall6s-Deffner; Meynell; Prigge 6 ; Roulet and Brenner; Seibert, Figueroa and Dufour; Takeda et al.; see chapter IV: "Immuno chemistry of the tubercle bacillus"). The question of how far these immune reactions can be used for the purpose of a protective vaccination has been taken up again only in recent years. Nevertheless, the interesting experiments of Buu-Hoi and Jouin, mentioned above, were undertaken to settle the question of whether the fatty acids under study were capable, after combining with protein, of protecting experimental animals against an infection with t~bercle bacilli. Further, Prigge5 has reported that the average length of survival after infection with small numbers of virulent tubercle bacilli is somewhat greater in guinea-pigs pre-treated with large doses of the TMS isolated by Seibert, Pedersen and Tiselius from culture filtrates of tubercle bacilli, than in control animals not pretreated. Raffel 2 has achieved no protective effect with TMS, apparently as a result of inadequate dosage. As after BCG vaccination, the protective effect appears only in a prolongation of life of limited duration, even with administration of very large quantities of TMS; and it would be subject to an increase (conformably with the conception presented in chapter V, according to which immunity against tuberculosis is to be interpreted as the resultant of the allergies towards all the antigens produced by the tubercle bacillus) only if, in addition to the tubercle bacillary protein, other immunogenic compounds were employed. Simultaneously, it was established that the experimental animals acquired a true anaphylaxis through TMS, in agreement with the

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statements of the American authors. On the contrary, as Raffel 2 has shown, reactions of the "tuberculin" type cannot be induced in the skin of animals made hypersensitive by TMS, either with TMS or with old-tuberculin, whereas they can be obtained in tuberculous guinea-pigs not only with old-tuberculin but also with TMS (Prigge 3 ). We attribute significance to these observations, particularly in view of the AO vaccine material discussed in chapter VI (3), whose mode of action is conceived by the originators of the preparation as reaction to the proteins produced by the tubercle bacillus. Even the sensitization attainable with TMS is, according to our conception, not identical with the sensitization against old-tuberculin; the antigens that induce the conversion must belong to different classes of substances. Up to now, attempts to utilize activators that have gfl.ined prominence in the preparation of highly active diphtheria vaccine materials (e.g. aluminum hydroxide) for the purpose of tuberculosis vaccination, seem to have led to no results-aside from the investigations of Ramon and his co-workers, and of Fritzsche. In recent years, the possibilities of an immunization with extracts of tubercle bacilli or partial antigens, have been subjected to renewed test. First of all Negre set up comparisons between his "methylantigen"an antigen prepared by methyl-alcohol extraction from tubercle bacilli dried and freed from fat by acetone-and BCG. The immunity developing after repeated injections of "methylantigen" occurs, according to Negre, earlier than that following BCG, is of shorter duration, and is accompanied by no tuberculin allergy. Of course, the last fact which, for purposes of definition, we would have to designate as "positive anergy," is to be evaluated most sceptically, if we take into account the possibility of a desensitization resulting from the cum ulative injections, and also the fact that the protective activity was measured not by the survival rate of the infected rabbits, but by the number of tuberculous lesions per animal. Seibert 11 too has investigated by animal experiment the proteins and polysaccharides isolated by her from culture filtrates of tubercle bacillus, for their immunizing activity; but she could not achieve, either with PPD-S or with the polysaccharides I or II, a protection equivalent even to that of BCG. Only the experiments on mice, carried out recently by Weiss and Dubos2 with a modified methylantigen in part activated by adjuvants, seem to have demonstrated a certain protective activity-measured by the prolongation of life of the vaccinated and infected mice; this was specified by the authors as of about the same order of magnitude

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as those following vaccination with killed pathogens or with living BCG. No absolute protection was attained here either. Youmans, Millman and Youmans achieved similar results, also with mice, using a vaccine prepared by ultra-centrifuging an extract from ground living tubercle bacilli, containing minute, non-acid-fast, enzymatically active particles which show certain characteristics of mitochondria. How far these particles are identical with the immunizing agents of the living tubercle bacillus is undecided. They might just as well be carriers of adsorptively linked substances which constitute the active principle. Nevertheless, the road opened by Youmans and co-workers, is undoubtedly worthy of further investigation. Of course, the findings presented by Dubos and Schaedler on heterologous increase of resistance in mice, after administration of BCG, counsel caution. Primarily, we must think also of participation by non-specific mechanisms (properdin system) in all investigations on increase of resistive powers against bacterial infection.

7. Summary and Outlook The problem of anti-tuberculosis vaccination is far indeed from a satisfactory solution. One of the most prominent experts on the matter, Bruno Lange 3, has characterized the situation as it existed up to his time, in the following words which are still pertinent even today (loc. cit., p. 326): "Thus, in the question of anti-tuberculosis vaccination, we have not so far gotten beyond experiments, these experiments are not very encouraging and, in the author's opinion, there exists, even in the future, only a faint prospect of achieving results in this way." As far as it concerns the research published up until now, we must concur with this opinion. It is valid even in regard to the results obtained with BCG. The optimism to which wide circles of the medical world have been tempted to yield (apparently in view of the necessity for an intensified fight against tuberculosis), we cannot consider to be justified as yet on grounds of really careful analysis of the results so far obtained. We consider a definite verdict on the value of BCG to be premature, and consider it essential to first carry out vaccinations that would make possible a precise evaluation. The same thing applies to other methods of vaccination. Beyond this, however, we are unable to share the pessimism of the above-mentioned distinguished investigator. As we have seen, recent developments have provided us with theoretically and practically significant insights into the structure of the tubercle bacillus and the chemical nature of its component parts. We have also deepened our knowledge concerning the character of allergy and immunity towards the partial antigens produced by the tubercle bacillus. Nevertheless, these insights, as has been shown particularly in chapter VI (4), have as yet been unable to give a real impetus towards a solution of the problem of anti-tuberculosis vaccination.

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We stand here before an unexplored territory, the opening-up of which will seem rewarding only to him who defends the conviction that methods can be found which promise more favourable results than those that have already been achieved along nature's way for immunization against tubercle bacilli. In so doing, we must consider in particular the possibility that the quantitative relationship of the pathogenically significant building-blocks and of the antigenically active compounds in the living and killed tubercle bacillus, might be unfavourable for the goal sought. To focus clearly what this primarily implies, we should once again recall the example of active anti-lockjaw vaccination. Even survival from tetanus leaves behind no reliable defence against fresh exogenous infections and repeated attacks. Only when we went on to employing, for active immunization, very large quantities, such as never occur in the disease, of a metabolic product concentrated in culture filtrates of the tetanus bacillus-after preceding detoxification with carbon disulphide (Ehrlich) or formaldehyde (Lowenstein)-did we succeed in solving a problem, for the mastering of which nature had shown no way. The attempts to create an immunity against tuberculosis, in similar ways, are only at their beginning, since we still do not know enough about the antigenically active components of the tubercle bacillus and their quantitative relationship as occurring under natural conditions; and this is true above all because-in contrast to the tetanus bacillus-we are dealing with more than just one antigen. Nevertheless, the hope does not seem inordinate, that we may, in the future, succeed in bringing to solution a problem, in which we have, as of now, progressed little beyond the accomplishments of Robert Koch.

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Subject Index Allergy, 15 ff. bacterial, 17, 24, 25 duration of, 62, 75 and immunity, 41 ff., 64 partial, 40, 47, 64 passively transferable, 19 tuberculin, see Tuberculin tuberculous, 17, 24 and vaccination, 62 ff. Anergy, 45, 46 Antibodies, 9, 11, 16, 19, 42 Antigen, 16, 39 partial, 40, 47, 83 soluble, for vaccination, 82 ff. Arthus phenomenon, 16, 27 BCG, 52 ff. allergy, 53, 62 bacteriology of, 53 fatalities from, 56 harmlessness of, 55 stability of virulence, 53, 58, 59 state control of, 59 vaccination complications, 55 vaccination statistics, 64 Bovovaccin, 51 Cord factor, 36 Desensitization, 43 ff. Friedmann bacillus vaccine, 52 Haptens, 17 Hyperergy, 44 partial hyperergy, 47 see also Allergy Infection and immunity, 6

Immunity, 6 ff. and allergy, 41 ff. and antibodies, 9, 16 and cellular defence, 11, 18 and resistance, 13 and tuberculin reaction, 12 Killed vaccines, 77 ff. activated, 78 ff. Anatuberculina lntegrale Petragnani, 81 AO, 79 ultra-violet-irradiated, 79 Koch's phenomenon, 6, 18 Mycolic acids, 33 ff. Phthionic acid, 30 ff. PPD, 26 Resistance, 3 ff. and immunity, 13 Shock, 15 Tauruman, 50 "Tb 18," 51 Tubercle-bacillus fats, 28 ff. lipoids, 28 ff. phosphatides, 30 ff. polysaccharides, 37 ff. proteins, 23 ff. and allergy, 27 fractionation of, 26 tuberculin activity of, 27 waxes, 33 and allergy, 34, 36 Tuberculin, 15 and allergy, 16, 17, 62 ff. antibodies, 44

108

INDEX

BCG tuberculin, 63 cytolysis, 20 ff. inhibitors, 44 purified (GT), 26 Tuberculin reaction, 6, 17, 27, 34, 44, 62,

74

anaphylactic type, 27 latency of, 46 delayed type, 27 Tuberculo-stearic acids, 30 Tuberkulin-muttersubstanz (TMS), 23 ff.

Vaccination experiments, 50 ff. with attenuated pathogens, 50 ff. with BCG, 52 concurrent, 44 intracutaneous, 62 with killed pathogens, 77 ff. oral, 53, 62 subcutaneous, 62 with soluble antigens, 82 ff. with virulent pathogens, 49 ff. Vitaltuberkulin, 51 Vole bacillus, 52, 75 ff.