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BCG vaccination and experimental corneal tuberculosis in the mouse
Tubercle, Lond., (I957), 38, I75
BCG Vaccination and Experimental Corneal Tuberculosis in the Mouse By R O Y G O U L D I N G from the Department of Pharnmcology, Guy's Hospital Aledical School, London While BCG vaccination is intended to protect against tuberculous infections, it is strange that vaccines are so seldom examined experimentally for their power of conferring immunity; instead, they are appraised more often according to their antigenic potency. The experiments on antituberculous immunity in mice carried out by Schwabacher and Wilson (I937) and, later, by Swedberg (I95I) demanded for their interpretation a detailed statistical analysis. More definite results were obtained by Dubos, Pierce and Schaefer (x953) , but they found it necessary for this purpose to sacrifice their animals, remove the viscera aseptically, prepare tissue emulsions and estimate the viable tubercle bacilli contained therein by plate-count techniques, all of which proved tedious and not without .danger to personnel. Subsequently, Bloch and Segal (i955) claimed satisfactory findings when they relied on survival times which, they asserted,, gave figures 'less equivocal than the ones obtained by a n y other method'. There seemed scope, nevertheless, for trying out a different criterion by which to evaluate the potency of BCG vaccines. Accordingly, their influence on experimental tuberculous infections of the mouse cornea was examined. • Methods
and
Materials
Rees and Robson (i95o) showed that by using a standardized culture of Alyco. tuberculosis var. boris and introducing this intracorneally into adult albino mice, one eye being used in each animal, a keratitic lesion first made its appearance after about ten days, progressed in size to reach a m a x i m u m involving the greater p a r t of the cornea in twenty to thirty days, then regressed somewhat and finally attained a chronic state persisting almost indefinitely. U p o n inspection with a binocular microscope, × I7-5, such a lesion could be 'scored' at any particular t i m e by assessing the proportion of the corneal area involved, arbitrarily regarding the whole cornea as 3.0. For a group of, say, Io mice, the scores at each twiceweekly inspection could be averaged and then plotted against the time (in days) elapsing from the day of infection. By this means a characteristic curve could be drawn, which was reproducible from one experiment • to another with untreated animals. T h e systemic administration of effective antituberculous drugs like streptomycin, p-amino-salieylate and isoniazld to such mice was found to suppress the corneal disease to such an extent that the curves then constructed took a very different course from that of the controls. I t was reasoned that i f a BCG vaccine given systemically to mice would produce an immunity which penetrated to the cornea then this too might modify the corneal disease in a manner similar to that with the • lrugs already mentioned.
Vaccines StockBCG.- A stock strain of BCG derived from a culture obtained from the Statens Seruminstitut,
'Copenhagen, was maintained by weekty subeuhure in the liquid medium of Dubos and Davis, interspersed with plating on L6wensteln-Jensen slopes. A seven-day culture in the liquid medium
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TUBERCLE
was diluted as required from Io - ~ to xo - ~ of the original suspension, using as diluent 0"05 p e r cent T w e e n 80 in physiological saline. Vaccination was performed by injecting intravenously into t h e tail 0.2 ml. per m o u s e of the diluted suspension. Viable counts were not m a d e on every vaccine specimen but, by the m e t h o d of K n o x (I955) with semi-solid agar, u n d i l u t e d cultures at seven days gave figures of the order of 2-5-6.0 × xo~ organisms per ml. for this stock BCG. '3[odified' Stock BCG Vaccine.- For one experiment, a suspension of this B C G was diluted to xo - ~ a n d autoclaved at 2o lb. per square inch for twenty minutes. Tested for xqability, a sample o f this vaccine failed to show growth either in Dubos' liquid m e d i u m or on L 6 w e n s t e l n - J e m e n slopes w h e n incubated at 37 ° C. for eight weeks. A n o t h e r g r o u p of mice was vaccinated with a viable culture of the s a m e dilution, b u t was then treated over the succeeding four weeks with isoniazid in the diet at the rate of 0. 3 rag. per a n i m a l per day. • ~lyeo. tuberculosis var. boris: H 3 7 R a . - F o r comparison with BCG, a stock culture of 3Iyco. tuberculosis, var. boris a n d a n o t h e r of Myco. tuberculosis H 3 7 R a were also used as vaccines. T h e s e were all standardized before dilution to contain the same n u m b e r of organisms by wet weigllt. Copenhagen B C G . - A m p o u l e s of B C G prepared b y t h e Statens Seruminstitut, C o p e n h a g e n , a n d supplied for clinical use in this c o u n t r y were diluted in the s a m e w a y as the stock B C G cultures. O n e such specimen (batch n u m b e r x I56 ) was injected within three days o f p r e p a r a t i o n , ' a n o t h e r (batch n u m b e r I xel) within seven days a n d a third (of t h e s a m e batch) was stored in t h e d a r k for a further seven days in the laboratory refrigerator at 5 ° C. T h e dose per m o u s e was a g a i n o.2 ml. intravenously. D a t a on the viable counts of these two specimens at the time of preparation were kindly" supplied b y D r K n u d T o l d e r l u n d of t h e Statens Seruminstitut. T h e figures, obtained by c o u n t i n g colonies u p o n L 6 w e m t e i n - J e m e n ' s m e d i u m after incubation at 37 ° C., were 2.3o x xo~ a n d x.7 ° X Io 7 p e r ml. respectively. T h e y were thus very similar to those found by the seml-solid a g a r m e t h o d for t h e stock laboratory B C G vaccine used in the first experiments. Freeze-dried Vaccines.- A freeze-drled specimen of B C G vaccine (batch n u m b e r 467) prepared at the Institut Pasteur, Paris, was reconstituted as directed a n d similarly diluted, b u t the dose perm o u s e was o.i 5 ml. intravenously because the phial was stated to contain the equivalent o f i .o mg~ bacilli instead of the 0-75 rag. in the C o p e n h a g e n material. F u r t h e r freeze-dried specimens prepared in the Glaxo Laboratories (batch n u m b e r 77) were reconstituted with Sauton's m e d i u m containing I/4,ooo w.v. T r i t o n W . R . 1339 w h i c h h a d been used for culturing the vaccine organisms in the first place. W i t h this s a m e fluid, dilutions were m a d e from to - z to Io - 6 . Viable counts of the u n d i l u t e d reconstituted suspension were again of" a similar order to the stock B C G culture (2.5-IO. 5 × Ion p e r ml.). T h e dose of the diluted vaccine was o-i 5 ml. per m o u s e intravenously. Controls.- T h e control mice in each experiment were injected intravenously at the s a m e time with the corresponding q u a n t i t y of diluent alone.
Animals A d u l t albino mice each weighing a b o u t ~5 g. were used throughout. These mice were housed in. m e t a l cages, each c o m p a r t m e n t of w h i c h held 5 animals a n d IO were assigned to each t r e a t m e n t group. T h e diet was in the form of rat cubes, s u p p l e m e n t e d weekly with crushed oats a n d rollerdried milk. These, with water, were allowed ad libitum.
Challenge Infections Intracorneal inoculations with 2~I.yco. tuberculosis var. boris (Rees a n d Robson, x95o ) were madeat intervals r a n g i n g from one to twenty-eight weeks following vaccination. T h e progress of t h e ensuing corneal disease was then recorded by the average 'scores' for each group of animals, thesebeing g r a p h e d against time. I n addition, for one of the experiments with Glaxo vaccine, n a m e l y that with dilutions of I o to - 3 a n d I o - * , a duplicate set ofvaceinated mice in groups of 6 was challenged by 31yco. tuberculosi¢ var. boris given intravenously, each a n i m a l receiving o-2 ml. of a n u n d i l u t e d seven-day culture byw a y of the tail. Such a n i n o c u l u m was previously found in u n t r e a t e d mice to set u p generalized tuberculosis causing d e a t h within three to seven weeks. T h e sur~qval times in b o t h control a n d vaccinated groups were noted. T w o groups of mice were treated w i t h cortisone for the four weeks following the challenge corneal infection. O n e of these groups was unvaccinated a n d the other h a d been vaccinated four weeks. previously with the stock B C G culture diluted io - 4 . Cortisone was given subcutaneously as t h e acetate, 0. 5 rag. per m o u s e p e r day.
Results Stock B C G . -
With an interval of four weeks between vaccination and corneal c h a l l e n g e , v a c c i n e d i l u t i o n s o f I o - 3 , I o - 4 a n d IO - 5 b r o u g h t a b o u t c o m p l e t e s u p pression of the corneal disease, while I o -2 was unexpectedly found to be only partially suppressive as compared with the controls. When the vaccine was further diluted,.
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Fro. x . - S h o w i n g the course of experimental corneal tuberculosis in mice vaccinated four weeks previously with a B C G culture given intravenously. Average size of lesions: unvaccinated controls (o o), B C G culture diluted zo - 4 ( A ~), B C G culture diluted lo - 5 ($k ~k), B C G culture diluted Io - e ( x . . . . × ) , B C G culture diluted xo-~ (0 ....
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Fro. 2. - Showing the course of experimental corneal tuberculosis in mice induced at different intervals following vaccination with a culture of B C G given intravenously. Average size of lesions: unvaccinated controls ( 1 1 e ) , interval of one week (O O), interval of two weeks ( X × ), interval of four weeks ($k & ) , interval of six weeks ( e . . . . e ) , interval of eight weeks (z~ . . . . A ) , interval of twenty-eight wee-ks)o . . . . o ) .
namely from lO -4 to Io -7, the degree of suppression was related inversely to tile dilution, xo -4 being almost complete in its effect and lower strengths progressively less so (Fig. I). With different intervals of time between vaccination and challenge, the vaccine diluted IO-a gave suppression that was slight but apparently significant after one week, greater after two weeks, complete at four and sLx weeks, a little less at eight weeks and further decreased but not entirely lost at twenty-elght weeks (Fig. ~). It was evident, therefore, that: (i) A BCG vaccine given intravenously to mice could set up an immunity response which infused the corneal tissues in such a way that subsequent experimental tuberculous infections in these tissues could be partially or completely suppressed. (ii) T h e degree of immunity developed was, in the main, directly related to the dose of vaccine administered. (iii) This immunity arose progressively following vaccination and, after reaching its maximum, slowly declined. Tile data under this heading corresponded closely with those of Bloch and Segal (i955). 'Modified' Vaccines.- In an experiment in which the stock vaccine dilution of Io -4 gave total protection against corneal infection four weeks later, the heat-treated, i.e. non-viable, vaccine of the same initial strength brought about such slight suppression that the curve was very little different from that for non-vaccinated controls. In the same experiment, the giving of isoniazid t o a n o t h e r group of mice between vaccination and challenge demonstrably reduced the immunity which might otherwise have developed, but failed to eliminate it completely (Fig. 3). Cortisone treatment of unvaccinated animals resulted in corneal disease more severe than in the controls. This had previously been reported by Naguib and Robson (I955). When cortisone was given similarly to vaccinated mice, not only did it dispose of the immunity which might otherwise have been present, but the corneal depredations were as advanced as those in the cortisone-treated mice which had not been vaccinated (Fig. 3)-
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Flo. 4--Showing the course of experimental corneal tuberculosis in mice vaccinated four weeks previously x~th cultures of Myco. tuberculosis,var. boris, BCG and Myco. tuberculosis,H37Ra. Average size of lesions: controls (e e), bovine diluted io -~ ((9 (9), bovine diluted io -5 ( × - - - - × ) , BCG diluted io -4 (A A), BCG diluted xo-5 (A -A), H37Ra dilutedjo-a (O O),H37Ra diluted lo -5 (× . . . . ×).
Myco. tuberculosis var. bovis; BCG; H37Ra.- As a vaccine, the bovine strain o f organisms used in dilutions of IO- 4 a n d Io -5, such doses b e i n g themselves n o n - l e t h a l , achieved complete protection from s u b s e q u e n t corneal infections. B C G was a g a i n f o u n d to be substantially protective at i 0 - 4 , b u t o n l y p a r t i a l l y so at io -5 a n d H 3 7 R a b e h a v e d m u c h like BCG, yet even more effectively (Fig. 4)Copenhagen BCG Vaccbze.- W i t h C o p e n h a g e n B C G used w i t h i n seven days o f p r e p a r a t i o n , prior v a c c i n a t i o n b r o u g h t a b o u t some protection against e x p e r i m e n t a l corneal infections m a d e four weeks later, m a x i m u m w i t h the xo -z dilution, n e a r l y complete with 1o -3, less with Io - 4 a n d nil with ]o - s (Fig. 5). Storing this v a c c i n e i n a refrigerator for a further week before its a d m i n i s t r a t i o n resulted in a corresponding deterioration in its protective capacity at all dilutions. Freeze-dried Vaccines.- I n a third e x p e r i m e n t in w h i c h C o p e n h a g e n vaccine used w i t h i n three days of p r e p a r a t i o n was c o m p a r e d with corresponding doses of reconstituted Pasteur freeze-dried vaccine, the C o p e n h a g e n material b e h a v e d as before b u t the freeze-dried specimen was less effective. T h e results with the Glaxo freeze-dried p r o d u c t are shown in Figs. 6, 7, a n d 8. Dilutions of zo -z, Io - a a n d Io -4 were wholly protective against e x p e r i m e n t a l corneal tuberculous infections, b u t less d r a m a t i c a l l y so against e x p e r i m e n t a l systemic infections. Survival times after systemic infections were a little prolonged i n those mice v a c c i n a t e d with the zo -z dilution, b u t scarcely different from the controls in those mice w h i c h were given the IO -3 a n d xo - 4 dilutions. I n d e e d , if a n y difference at all can be perceived b e t w e e n the last two groups it is to suggest t h a t the latter was more effective t h a n t h e former. Similar inconclusive d a t a were collected b y M u g g l e t o n (personal
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communication) when he gave the same vaccine to mice and later challenged them by intravenous inoculations of virulent tubercle bacilli. Repeating the corneal experiments with further dilutions of vaccine, namely lO -4 , lO -5 and IO -6 , there was complete protection with the first two of these doses and considerable, albeit partial, protection with the third and lowest of these doses, despite the fact - if the viable counts are to be taken literally-that the estimated number of BCG organisms introduced into each animal w a s n o more than IOO, IO and i respectively for the three vaccine dilutions. Discussion
T h e experiments now reported confirm what Dubos and others (I953) and other workers have already described, that resistance towards tuberculous infections can be induced in mice b y the introduction of living B C G and other strains of tuberculous organisms. Furthermore, whatever the nature of the immunity factor, it seems to penetrate to the cornea as readily as to other parts of the body. I t develops gradually, reaching a m a x i m u m at about four weeks, remains thus for a few weeks more and then wanes slowly but not completely by t~venty-eight weeks. T h e degree of protection attained is related directly to the dose of vaccine employed, except that a particularly high dose m a y be somewhat less potent than those rather lower. This technique m a y accordingly provide a simple method by which the potency of BCG vaccines might be estimated experimentally, in a comparatively short time. T h e diminished immunity in those mice treated with isoniazid after vaccination is in accord with the findings of Dubos and others (I953) but not with those of Bloch and Segal (I955). I t is possible that the B C G organisms used as vaccine by the former were more susceptible to the drug in vi~'o than the R I R v used by the latter. There is reason to believe that isoniazid at the rate of 0. 3 mg. per mouse per day would effectively counter the proliferation of as few as a thousand BCG organisnls comprising the vaccine dose in the present experiments, without obliterating them entirely. A few would p r o b a b l y survive to create that low level of immunity which was afterwards observed. Cortisone apparently lowered the resistance to infection so profoundly that the benefit otherwise derived from vaccination, always incomplete, was completely overwhelmed. T h e tests with different strains of tubercle bacilli were intended to throw some light on the relationship of immunity to the multiplication of the vaccine organisms within the host. Those of the virulent bovine strain are known to proliferate and glve rise to conspicuous lesions in the tissues, the attenuated BCG proliferate to a limited extent, but the avirulent H 3 7 R a are alleged not to do so at all. O n the other hand, the tissue changes provoked by H 3 7 R a m a y be as great or, according to Rist (I955) , even greater, than those caused by the latter. Perhaps, after all, it is not the activity of the vaccine organisms per se upon which the generation of immunity depends but, instead, the cellular changes in the host to which they give rise. This m a y also explain why the non-viable, i.e. heat-treated, BCG vaccine in the same dose was totally ineffective prophylactically, for in this state and in this quantity it provoked no host reactions. Finally, with the freeze-dried BCG vaccines, the evidence points to the level of immunity being related proportionately to the n u m b e r of viable bacilli injected. So the experimental intracorneal challenge m a y achieve no more eventually than a viable count of the original specimen by a circuitous means or, possibly, the viable potential ultimately attained in the mouse body. At least, the experimental observation that some immunity is developed by the presence of no more than I BCG organism throws doubt on the ultimate (though not necessarily the comparative) accuracy of viable counts undertaken hz vitro. As a laboratory method, the intracorneal technique nevertheless lends itself conveniently to studies on anti-tuberculous immunity. Whether, in more practical
BCG VACCINATION
IN MICE
I8I
circumstances, it can be adopted as a means o f testing and standardizing commercial vaccines can be determined in the long r u n only b y correlating over the years the efficacy of different batches clinically with their performance previously in the laboratory. Considering h o w long it has taken to collect convincing evidence from clinical trials to answer the single question w h e t h e r B C G is of a n y value at all, the rigours and tribulations o f a further follow-up o f this kind m i g h t be formidable enough to deter.even the most ambitious investigator.
Summary By giving viable B C G organisms intravenously to mice an i m m u n i t y has been set up which protects against subsequent corneal tuberculosis experimentally induced. T h e degree of protection has been quantitatively related to the dose o f vaccine employed. This i m m u n i t y takes a b o u t four weeks to reach its m a x i m u m and remains thus for a few weeks a n d later diminishes gradually but is not lost altogether by twentyeight weeks. A heat-killed vaccine o f the same dosage has proved ineffective, while treating the m!ce with isoniazid after vaccination has considerably reduced t h e level o f : m m u n i t y from that otherwise expected. Cortisone, too, given after the challenge corneal infection, has overcome the vaccination immunity. A comparison o f similar doses o f bovine, B C G and H 3 7 R a strains of tubercle bacilli as vaccines has shown the first of these to be the most potent, with the other ~vo of a similar order o f potency. Reconstituted freeze-dried B C G vaccines have also been satisfactory for establishing i m m u n i t y in mice, their capacities in this respect being a p p a r e n t l y determined b y the n u m b e r o f viable organisms contained therein. T h e possibility of using the intracorneal technique for estimating the p o t e n c y o f antituberculosis vaccines is considered. I am very grateful to Professor J. M. Robson for the facilities and encouragement he has afforded me, to Professor G. Payllng Wright for helpful discussions and to Mrs D. Smith for her invaluable and conscientious assistance throughout. I am also indebted to all those people who so readily answered my requests for vaccine specimens, particularly to Professor R. Knox and Dr D. Chalmers of Guy's Hospital Medical School, Colonel Bensted of the Central Publle Health Laboratories and Dr. J. Ungar and Dr P. W. Muggleton of Gla-xo Laboratories Ltd. The work described in this paper formed part of a thesis accepted by the University of London for the degree of Doctor of Medicine.
References Bloch, H., and Segal, W. (1955) Amer. Rev. Tuberc., 7 x, 228. Dubos, R.J., Pierce, C. H., and Schaefer, W. B. (x953)~J. exp. Med., 97, 207Knox, R. (1955) Lancet, ii, I I o. Naguib, M., and Robson, J. M. (I955) Brit. J. Pharmacol., xo, 387. Rees, R.J.W., and Robson, J. M. 0950) Brit. J. Pharmacol., 5, 77Rist, N. (1955) In Ciba Foundation Symposium on Experimental Tuberculosis, ed. b y G. E. Wolstenholme and M. P. Cameron, p. 243, Churchill, London. Schwabacher, H., and Wilson, G. S. (1937) Tubercle., Lond., x8, 49~. Swedberg, B. 095Q Acta reed. seand., Suppl. 254.
O
BCG Vaccination and Experimental Corneal Tuberculosis in the Mouse By R O Y G O U L D I N G from the Department of Pharnmcology, Guy's Hospital Aledical School, London While BCG vaccination is intended to protect against tuberculous infections, it is strange that vaccines are so seldom examined experimentally for their power of conferring immunity; instead, they are appraised more often according to their antigenic potency. The experiments on antituberculous immunity in mice carried out by Schwabacher and Wilson (I937) and, later, by Swedberg (I95I) demanded for their interpretation a detailed statistical analysis. More definite results were obtained by Dubos, Pierce and Schaefer (x953) , but they found it necessary for this purpose to sacrifice their animals, remove the viscera aseptically, prepare tissue emulsions and estimate the viable tubercle bacilli contained therein by plate-count techniques, all of which proved tedious and not without .danger to personnel. Subsequently, Bloch and Segal (i955) claimed satisfactory findings when they relied on survival times which, they asserted,, gave figures 'less equivocal than the ones obtained by a n y other method'. There seemed scope, nevertheless, for trying out a different criterion by which to evaluate the potency of BCG vaccines. Accordingly, their influence on experimental tuberculous infections of the mouse cornea was examined. • Methods
and
Materials
Rees and Robson (i95o) showed that by using a standardized culture of Alyco. tuberculosis var. boris and introducing this intracorneally into adult albino mice, one eye being used in each animal, a keratitic lesion first made its appearance after about ten days, progressed in size to reach a m a x i m u m involving the greater p a r t of the cornea in twenty to thirty days, then regressed somewhat and finally attained a chronic state persisting almost indefinitely. U p o n inspection with a binocular microscope, × I7-5, such a lesion could be 'scored' at any particular t i m e by assessing the proportion of the corneal area involved, arbitrarily regarding the whole cornea as 3.0. For a group of, say, Io mice, the scores at each twiceweekly inspection could be averaged and then plotted against the time (in days) elapsing from the day of infection. By this means a characteristic curve could be drawn, which was reproducible from one experiment • to another with untreated animals. T h e systemic administration of effective antituberculous drugs like streptomycin, p-amino-salieylate and isoniazld to such mice was found to suppress the corneal disease to such an extent that the curves then constructed took a very different course from that of the controls. I t was reasoned that i f a BCG vaccine given systemically to mice would produce an immunity which penetrated to the cornea then this too might modify the corneal disease in a manner similar to that with the • lrugs already mentioned.
Vaccines StockBCG.- A stock strain of BCG derived from a culture obtained from the Statens Seruminstitut,
'Copenhagen, was maintained by weekty subeuhure in the liquid medium of Dubos and Davis, interspersed with plating on L6wensteln-Jensen slopes. A seven-day culture in the liquid medium
I76
TUBERCLE
was diluted as required from Io - ~ to xo - ~ of the original suspension, using as diluent 0"05 p e r cent T w e e n 80 in physiological saline. Vaccination was performed by injecting intravenously into t h e tail 0.2 ml. per m o u s e of the diluted suspension. Viable counts were not m a d e on every vaccine specimen but, by the m e t h o d of K n o x (I955) with semi-solid agar, u n d i l u t e d cultures at seven days gave figures of the order of 2-5-6.0 × xo~ organisms per ml. for this stock BCG. '3[odified' Stock BCG Vaccine.- For one experiment, a suspension of this B C G was diluted to xo - ~ a n d autoclaved at 2o lb. per square inch for twenty minutes. Tested for xqability, a sample o f this vaccine failed to show growth either in Dubos' liquid m e d i u m or on L 6 w e n s t e l n - J e m e n slopes w h e n incubated at 37 ° C. for eight weeks. A n o t h e r g r o u p of mice was vaccinated with a viable culture of the s a m e dilution, b u t was then treated over the succeeding four weeks with isoniazid in the diet at the rate of 0. 3 rag. per a n i m a l per day. • ~lyeo. tuberculosis var. boris: H 3 7 R a . - F o r comparison with BCG, a stock culture of 3Iyco. tuberculosis, var. boris a n d a n o t h e r of Myco. tuberculosis H 3 7 R a were also used as vaccines. T h e s e were all standardized before dilution to contain the same n u m b e r of organisms by wet weigllt. Copenhagen B C G . - A m p o u l e s of B C G prepared b y t h e Statens Seruminstitut, C o p e n h a g e n , a n d supplied for clinical use in this c o u n t r y were diluted in the s a m e w a y as the stock B C G cultures. O n e such specimen (batch n u m b e r x I56 ) was injected within three days o f p r e p a r a t i o n , ' a n o t h e r (batch n u m b e r I xel) within seven days a n d a third (of t h e s a m e batch) was stored in t h e d a r k for a further seven days in the laboratory refrigerator at 5 ° C. T h e dose per m o u s e was a g a i n o.2 ml. intravenously. D a t a on the viable counts of these two specimens at the time of preparation were kindly" supplied b y D r K n u d T o l d e r l u n d of t h e Statens Seruminstitut. T h e figures, obtained by c o u n t i n g colonies u p o n L 6 w e m t e i n - J e m e n ' s m e d i u m after incubation at 37 ° C., were 2.3o x xo~ a n d x.7 ° X Io 7 p e r ml. respectively. T h e y were thus very similar to those found by the seml-solid a g a r m e t h o d for t h e stock laboratory B C G vaccine used in the first experiments. Freeze-dried Vaccines.- A freeze-drled specimen of B C G vaccine (batch n u m b e r 467) prepared at the Institut Pasteur, Paris, was reconstituted as directed a n d similarly diluted, b u t the dose perm o u s e was o.i 5 ml. intravenously because the phial was stated to contain the equivalent o f i .o mg~ bacilli instead of the 0-75 rag. in the C o p e n h a g e n material. F u r t h e r freeze-dried specimens prepared in the Glaxo Laboratories (batch n u m b e r 77) were reconstituted with Sauton's m e d i u m containing I/4,ooo w.v. T r i t o n W . R . 1339 w h i c h h a d been used for culturing the vaccine organisms in the first place. W i t h this s a m e fluid, dilutions were m a d e from to - z to Io - 6 . Viable counts of the u n d i l u t e d reconstituted suspension were again of" a similar order to the stock B C G culture (2.5-IO. 5 × Ion p e r ml.). T h e dose of the diluted vaccine was o-i 5 ml. per m o u s e intravenously. Controls.- T h e control mice in each experiment were injected intravenously at the s a m e time with the corresponding q u a n t i t y of diluent alone.
Animals A d u l t albino mice each weighing a b o u t ~5 g. were used throughout. These mice were housed in. m e t a l cages, each c o m p a r t m e n t of w h i c h held 5 animals a n d IO were assigned to each t r e a t m e n t group. T h e diet was in the form of rat cubes, s u p p l e m e n t e d weekly with crushed oats a n d rollerdried milk. These, with water, were allowed ad libitum.
Challenge Infections Intracorneal inoculations with 2~I.yco. tuberculosis var. boris (Rees a n d Robson, x95o ) were madeat intervals r a n g i n g from one to twenty-eight weeks following vaccination. T h e progress of t h e ensuing corneal disease was then recorded by the average 'scores' for each group of animals, thesebeing g r a p h e d against time. I n addition, for one of the experiments with Glaxo vaccine, n a m e l y that with dilutions of I o to - 3 a n d I o - * , a duplicate set ofvaceinated mice in groups of 6 was challenged by 31yco. tuberculosi¢ var. boris given intravenously, each a n i m a l receiving o-2 ml. of a n u n d i l u t e d seven-day culture byw a y of the tail. Such a n i n o c u l u m was previously found in u n t r e a t e d mice to set u p generalized tuberculosis causing d e a t h within three to seven weeks. T h e sur~qval times in b o t h control a n d vaccinated groups were noted. T w o groups of mice were treated w i t h cortisone for the four weeks following the challenge corneal infection. O n e of these groups was unvaccinated a n d the other h a d been vaccinated four weeks. previously with the stock B C G culture diluted io - 4 . Cortisone was given subcutaneously as t h e acetate, 0. 5 rag. per m o u s e p e r day.
Results Stock B C G . -
With an interval of four weeks between vaccination and corneal c h a l l e n g e , v a c c i n e d i l u t i o n s o f I o - 3 , I o - 4 a n d IO - 5 b r o u g h t a b o u t c o m p l e t e s u p pression of the corneal disease, while I o -2 was unexpectedly found to be only partially suppressive as compared with the controls. When the vaccine was further diluted,.
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Fro. x . - S h o w i n g the course of experimental corneal tuberculosis in mice vaccinated four weeks previously with a B C G culture given intravenously. Average size of lesions: unvaccinated controls (o o), B C G culture diluted zo - 4 ( A ~), B C G culture diluted lo - 5 ($k ~k), B C G culture diluted Io - e ( x . . . . × ) , B C G culture diluted xo-~ (0 ....
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Fro. 2. - Showing the course of experimental corneal tuberculosis in mice induced at different intervals following vaccination with a culture of B C G given intravenously. Average size of lesions: unvaccinated controls ( 1 1 e ) , interval of one week (O O), interval of two weeks ( X × ), interval of four weeks ($k & ) , interval of six weeks ( e . . . . e ) , interval of eight weeks (z~ . . . . A ) , interval of twenty-eight wee-ks)o . . . . o ) .
namely from lO -4 to Io -7, the degree of suppression was related inversely to tile dilution, xo -4 being almost complete in its effect and lower strengths progressively less so (Fig. I). With different intervals of time between vaccination and challenge, the vaccine diluted IO-a gave suppression that was slight but apparently significant after one week, greater after two weeks, complete at four and sLx weeks, a little less at eight weeks and further decreased but not entirely lost at twenty-elght weeks (Fig. ~). It was evident, therefore, that: (i) A BCG vaccine given intravenously to mice could set up an immunity response which infused the corneal tissues in such a way that subsequent experimental tuberculous infections in these tissues could be partially or completely suppressed. (ii) T h e degree of immunity developed was, in the main, directly related to the dose of vaccine administered. (iii) This immunity arose progressively following vaccination and, after reaching its maximum, slowly declined. Tile data under this heading corresponded closely with those of Bloch and Segal (i955). 'Modified' Vaccines.- In an experiment in which the stock vaccine dilution of Io -4 gave total protection against corneal infection four weeks later, the heat-treated, i.e. non-viable, vaccine of the same initial strength brought about such slight suppression that the curve was very little different from that for non-vaccinated controls. In the same experiment, the giving of isoniazid t o a n o t h e r group of mice between vaccination and challenge demonstrably reduced the immunity which might otherwise have developed, but failed to eliminate it completely (Fig. 3). Cortisone treatment of unvaccinated animals resulted in corneal disease more severe than in the controls. This had previously been reported by Naguib and Robson (I955). When cortisone was given similarly to vaccinated mice, not only did it dispose of the immunity which might otherwise have been present, but the corneal depredations were as advanced as those in the cortisone-treated mice which had not been vaccinated (Fig. 3)-
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Flo. 4--Showing the course of experimental corneal tuberculosis in mice vaccinated four weeks previously x~th cultures of Myco. tuberculosis,var. boris, BCG and Myco. tuberculosis,H37Ra. Average size of lesions: controls (e e), bovine diluted io -~ ((9 (9), bovine diluted io -5 ( × - - - - × ) , BCG diluted io -4 (A A), BCG diluted xo-5 (A -A), H37Ra dilutedjo-a (O O),H37Ra diluted lo -5 (× . . . . ×).
Myco. tuberculosis var. bovis; BCG; H37Ra.- As a vaccine, the bovine strain o f organisms used in dilutions of IO- 4 a n d Io -5, such doses b e i n g themselves n o n - l e t h a l , achieved complete protection from s u b s e q u e n t corneal infections. B C G was a g a i n f o u n d to be substantially protective at i 0 - 4 , b u t o n l y p a r t i a l l y so at io -5 a n d H 3 7 R a b e h a v e d m u c h like BCG, yet even more effectively (Fig. 4)Copenhagen BCG Vaccbze.- W i t h C o p e n h a g e n B C G used w i t h i n seven days o f p r e p a r a t i o n , prior v a c c i n a t i o n b r o u g h t a b o u t some protection against e x p e r i m e n t a l corneal infections m a d e four weeks later, m a x i m u m w i t h the xo -z dilution, n e a r l y complete with 1o -3, less with Io - 4 a n d nil with ]o - s (Fig. 5). Storing this v a c c i n e i n a refrigerator for a further week before its a d m i n i s t r a t i o n resulted in a corresponding deterioration in its protective capacity at all dilutions. Freeze-dried Vaccines.- I n a third e x p e r i m e n t in w h i c h C o p e n h a g e n vaccine used w i t h i n three days of p r e p a r a t i o n was c o m p a r e d with corresponding doses of reconstituted Pasteur freeze-dried vaccine, the C o p e n h a g e n material b e h a v e d as before b u t the freeze-dried specimen was less effective. T h e results with the Glaxo freeze-dried p r o d u c t are shown in Figs. 6, 7, a n d 8. Dilutions of zo -z, Io - a a n d Io -4 were wholly protective against e x p e r i m e n t a l corneal tuberculous infections, b u t less d r a m a t i c a l l y so against e x p e r i m e n t a l systemic infections. Survival times after systemic infections were a little prolonged i n those mice v a c c i n a t e d with the zo -z dilution, b u t scarcely different from the controls in those mice w h i c h were given the IO -3 a n d xo - 4 dilutions. I n d e e d , if a n y difference at all can be perceived b e t w e e n the last two groups it is to suggest t h a t the latter was more effective t h a n t h e former. Similar inconclusive d a t a were collected b y M u g g l e t o n (personal
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TUBERCLE
communication) when he gave the same vaccine to mice and later challenged them by intravenous inoculations of virulent tubercle bacilli. Repeating the corneal experiments with further dilutions of vaccine, namely lO -4 , lO -5 and IO -6 , there was complete protection with the first two of these doses and considerable, albeit partial, protection with the third and lowest of these doses, despite the fact - if the viable counts are to be taken literally-that the estimated number of BCG organisms introduced into each animal w a s n o more than IOO, IO and i respectively for the three vaccine dilutions. Discussion
T h e experiments now reported confirm what Dubos and others (I953) and other workers have already described, that resistance towards tuberculous infections can be induced in mice b y the introduction of living B C G and other strains of tuberculous organisms. Furthermore, whatever the nature of the immunity factor, it seems to penetrate to the cornea as readily as to other parts of the body. I t develops gradually, reaching a m a x i m u m at about four weeks, remains thus for a few weeks more and then wanes slowly but not completely by t~venty-eight weeks. T h e degree of protection attained is related directly to the dose of vaccine employed, except that a particularly high dose m a y be somewhat less potent than those rather lower. This technique m a y accordingly provide a simple method by which the potency of BCG vaccines might be estimated experimentally, in a comparatively short time. T h e diminished immunity in those mice treated with isoniazid after vaccination is in accord with the findings of Dubos and others (I953) but not with those of Bloch and Segal (I955). I t is possible that the B C G organisms used as vaccine by the former were more susceptible to the drug in vi~'o than the R I R v used by the latter. There is reason to believe that isoniazid at the rate of 0. 3 mg. per mouse per day would effectively counter the proliferation of as few as a thousand BCG organisnls comprising the vaccine dose in the present experiments, without obliterating them entirely. A few would p r o b a b l y survive to create that low level of immunity which was afterwards observed. Cortisone apparently lowered the resistance to infection so profoundly that the benefit otherwise derived from vaccination, always incomplete, was completely overwhelmed. T h e tests with different strains of tubercle bacilli were intended to throw some light on the relationship of immunity to the multiplication of the vaccine organisms within the host. Those of the virulent bovine strain are known to proliferate and glve rise to conspicuous lesions in the tissues, the attenuated BCG proliferate to a limited extent, but the avirulent H 3 7 R a are alleged not to do so at all. O n the other hand, the tissue changes provoked by H 3 7 R a m a y be as great or, according to Rist (I955) , even greater, than those caused by the latter. Perhaps, after all, it is not the activity of the vaccine organisms per se upon which the generation of immunity depends but, instead, the cellular changes in the host to which they give rise. This m a y also explain why the non-viable, i.e. heat-treated, BCG vaccine in the same dose was totally ineffective prophylactically, for in this state and in this quantity it provoked no host reactions. Finally, with the freeze-dried BCG vaccines, the evidence points to the level of immunity being related proportionately to the n u m b e r of viable bacilli injected. So the experimental intracorneal challenge m a y achieve no more eventually than a viable count of the original specimen by a circuitous means or, possibly, the viable potential ultimately attained in the mouse body. At least, the experimental observation that some immunity is developed by the presence of no more than I BCG organism throws doubt on the ultimate (though not necessarily the comparative) accuracy of viable counts undertaken hz vitro. As a laboratory method, the intracorneal technique nevertheless lends itself conveniently to studies on anti-tuberculous immunity. Whether, in more practical
BCG VACCINATION
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circumstances, it can be adopted as a means o f testing and standardizing commercial vaccines can be determined in the long r u n only b y correlating over the years the efficacy of different batches clinically with their performance previously in the laboratory. Considering h o w long it has taken to collect convincing evidence from clinical trials to answer the single question w h e t h e r B C G is of a n y value at all, the rigours and tribulations o f a further follow-up o f this kind m i g h t be formidable enough to deter.even the most ambitious investigator.
Summary By giving viable B C G organisms intravenously to mice an i m m u n i t y has been set up which protects against subsequent corneal tuberculosis experimentally induced. T h e degree of protection has been quantitatively related to the dose o f vaccine employed. This i m m u n i t y takes a b o u t four weeks to reach its m a x i m u m and remains thus for a few weeks a n d later diminishes gradually but is not lost altogether by twentyeight weeks. A heat-killed vaccine o f the same dosage has proved ineffective, while treating the m!ce with isoniazid after vaccination has considerably reduced t h e level o f : m m u n i t y from that otherwise expected. Cortisone, too, given after the challenge corneal infection, has overcome the vaccination immunity. A comparison o f similar doses o f bovine, B C G and H 3 7 R a strains of tubercle bacilli as vaccines has shown the first of these to be the most potent, with the other ~vo of a similar order o f potency. Reconstituted freeze-dried B C G vaccines have also been satisfactory for establishing i m m u n i t y in mice, their capacities in this respect being a p p a r e n t l y determined b y the n u m b e r o f viable organisms contained therein. T h e possibility of using the intracorneal technique for estimating the p o t e n c y o f antituberculosis vaccines is considered. I am very grateful to Professor J. M. Robson for the facilities and encouragement he has afforded me, to Professor G. Payllng Wright for helpful discussions and to Mrs D. Smith for her invaluable and conscientious assistance throughout. I am also indebted to all those people who so readily answered my requests for vaccine specimens, particularly to Professor R. Knox and Dr D. Chalmers of Guy's Hospital Medical School, Colonel Bensted of the Central Publle Health Laboratories and Dr. J. Ungar and Dr P. W. Muggleton of Gla-xo Laboratories Ltd. The work described in this paper formed part of a thesis accepted by the University of London for the degree of Doctor of Medicine.
References Bloch, H., and Segal, W. (1955) Amer. Rev. Tuberc., 7 x, 228. Dubos, R.J., Pierce, C. H., and Schaefer, W. B. (x953)~J. exp. Med., 97, 207Knox, R. (1955) Lancet, ii, I I o. Naguib, M., and Robson, J. M. (I955) Brit. J. Pharmacol., xo, 387. Rees, R.J.W., and Robson, J. M. 0950) Brit. J. Pharmacol., 5, 77Rist, N. (1955) In Ciba Foundation Symposium on Experimental Tuberculosis, ed. b y G. E. Wolstenholme and M. P. Cameron, p. 243, Churchill, London. Schwabacher, H., and Wilson, G. S. (1937) Tubercle., Lond., x8, 49~. Swedberg, B. 095Q Acta reed. seand., Suppl. 254.
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