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Some biological, biochemical and morphological changes and their correlations in selected BCG vaccine strains
ffournal of Biological ~tandardizat~n 1976 4, 319--327
S o m e bi morphol co elatio
cal, biochemic and al ch ges and their in selected B C G vaccine
.7. Pr:tchov~,t .~. 2d~ra,$ H . ~/loheIsk~,§ Z. ~gr§ and ff. Galliovd§
Changes m the biolo~caI, bloehem_i~ and morphological properties of three BeG s~ns, C o p e n h a g e n B C G 1331 a n d ~e¢o o f its d ~ v a t l v e s , P r a b ~ B C G 725 S H a n d P r a h a B C G 725 S A S , w e r e s t u d i e d a n d . T h e s e c h a n g e s w e r e c a u s e d b y c h e m i c a l (cult~.Ire medga) and p h y s i ~ factors (fre~z~-drying). T h e highest i.m_rnvJaizingl~t~ncy ~'~d the Im'ghest lipid content were displayed, both prior to and after ~reeze ch-ying, by the Da~_ish s~. cultivation of the Danish str-,dnon different media fin Prague resulted in a d e c r e a s e o f its p o t e n c y a n d lipid c o n t e n t . B o t h o f t h e C z e c h o s l o v a k s t r a i n s d e r i v e d in this m a n n e r , B C G 725 S H a n d B C G 725 S A S , d i f f e r e d m u t u a l l y b y t h e / r L m m t m l z i n g p o t e n c y , llpid c o n t e n t a n d o f L n t a ~ c~LLs. T h e i r r ' ~ a n i z i n g p o t e n c y a n d l i p i d c o n t e n t o f t h e s e t w o s t r a i n s w e r e m ~ v e r s e p r o p o r t i o n ; so w e r e t h e irnrnmnlzing potency and percenta~ of inta~ ria. The expe~mts s u g g e s t e d t h a t s u b s t a n t i a l c h a n g e s d e v e l o p a f t e r p h y s i c a l a c t i o n (freezed-~J;mg) only in stre~as being tx-~q_int~ed on f i ~ cultttre m e ~ F r ~ z e ~ r l n g changed the relationship betwedn Lng p o t e n ~ s-~d Hpld content into a direct one in both of the Czechoslovak straJ~ns. T~ne re~,ionship betweext ~ t m A z flag potenc~l and percentage of Lstact mycobactefia remained inverse. In B e G 72$ S H , total Hpids a n d L t r m a t m ~ g p o t e n c y a n d t h e percem-tag¢ o f k-~tact cells i n c r e a s e d a~er__~-eeze d r y i n g . I ~ B C G 725 S A S o n t h e o t h e r h a t t d t h e i r m x a u n ~ g poten~ fincreased, llpids z e m ~ e d o n a s t a b l e level a n d t h e o f L-tract cells dec~-~ased. T h e substant/al changes in t h e s e p induced by" Jfireeze-dt~Jng s u b s e q u e n t l y r e t u r n e d to t h e originaI v a l u e s i r r e s p e c t i v e o f w h e t h e r t h e strai_jxs h a d u n d e r g o n e a n u m b e r o f freeze-dr~_.ng p r o c e d u r e s o r h a d b e e n p a s s a g e d d i r e c t l y a f t e r a sfingle freeze-d-_~'ing v a n . Aza essential effect w a s t h u s o n l y p r o d u c e d b y t h e first f r e - e z e - ~ g , a n d t h e e.h~sagea ap//ear n o t t o b e stable. * R e c e i v e d f o r p u b l i c a t i o n 20 A p ~ 1976. f S t a t e I r ~ t i t u t e f o r D r u g C o n t r o l ( D i r . J; B~CL~ek, M . D . C . S c . ) , P r a g u e . ! s d H c r o b i o l o # M I n s t i t u t e , }J~_dlcal FacuIg¢, C h a r l e s U n i v e r s i W ( H e a d A s s i s t a n t P r o f e s s o r John, M.D.C.Sc.) Prague. § Institaate o f H y g i e n e a n d E p i (Dh-. P r o f e s s o r F . J a n d a , M . D . D . S c . ) , P r a g u e .
319
J . P R UoC H O
V
A, M . M A R A
H. I'vIOHELSKA, Z. ~IR A N D
J. G A L L I O V A
INTRODUCTION Although there has been a considerable decrease in t u ~ r c u l o s i s morbidity in Czechoslovakia, ha p a r t i c u l ~ among chiIdren and adolescents, prevention of a disease o f such consequence is still indispensable. T h e most i m p o r t a n t component of T B prevention is B C G vaccination. T h e original French B C G sti~in has, in the course of its long-term maintenance u n d e r varying cultivation conditions in different laboratories, undergone some substantial biological, b i o c h e m i ~ and m o r p h o l o ~ c a l changes. T h e selection of a variant of the strain and cultivation s that w ~ r a n t standard properties and t~igh vaccine effectiveness thus becomes a primary problem. Ac ~ e present steady was especially mined at investigating chmnges in the b i o l o g i ~ properties o f vaccine sh-ains of which the most important is Lrnmmaizing potency. AdditionaI aims were to explore, in a model experiment, w h e t h e r there were any concrete relations between the changes concerned (biological, biochemical and morphological) and to estimate the changes quantitatively. MATERIAL
AND
METHODS
Strains and cultivation Danish vaccine prepared from strain B C G 1331 was used as a standard. T h i s strain was pa~saged in Japan on bile-potato, then on asparagine-contaLaing original Sauton's m e d i u m and t h e n freeze-dried. On the basis of our l o n g - t e ~ experiences with this standard strain, the calculated percentage of immunizing potency o f the other s t r ~ s was related statistically 4o its Lrrm~unizing potency. T h e actual study was concerned with the Czechoslovak vaccine strah~ B C G 725, which h a d been derived from batch 725 of the Danish s ~ a i n in 1946. Since 1947 it has been passaged first on Sauton's potato and then d~ectly in Sauton's m e d i u m in which asparagine, has been replaced by e n ~ S c hydrolysate o f easeLn (SH medium). I n addition, the same B C G 725 strain was also studied after maintenance on Sauton's potato and cultivation in o r i ~ n a l Sauton's m e d i u m (SAS m e , u r n ) , containLng ~ p a r a g i n e as the only amino acid.
Freeze-drying of BCG-strab,.s Freeze-dr-ying was performed on an L Z 9B apparatus (Frigera, Kolin, Czechoslovakia). ~ e oalt~are m e d i u m of 72day ~ I t u r e s was remoo~d in a Birghaug b a ~ e r i a l press. T h e bacterial mass was homogenized by shaking with stainless steel balls and diI * U ted with 1~o Na.glutamate to a basic sus1~znsion of 50 m g o f ~ m i d r y wt./mL T h i s suspension was further diluted with 1 % Na-glutmmate to 10 mg[m_l, d i s p e n ~ d in 0.5 ml p o ~ o n s into a m p u l e s and f r o w n in a deep freezer t o - - 4 0 °C for 3 h. T h e anapoules were then transferred into the freeze-drying apparat~as and freez~e-dried for 16-18 h. Using a semiautomatic Edwards appliance, ampoule necks were con~_ricted, and d ~ n g was completed on an Edwards secondary freeze-drier (16 h); ampoules were seaIed u n d e r vacuum.
A
t of model experiment
A B C G stnLLn that had n o t previously been f r e e ~ - d r i e d (Former) was (I) freeze-dried or ( 2 ) s u b j e c t e d to further p ~ a g i n g . Tile freeze-dried strain was seeded and again either (1) pa~aged or (2) freeze-dried r e p u t e d l y (Fig. 1). I n this way three lines were obtained: 320
CHANGES I N B C G
VACCINE STRAINS
(1) non-freeze-dried strain; (2) ~ n freeze-dried once; (3) strain f r e e ~ - d r i e d repeatedly. Since liquid vaccine prepared from non-freeze-dried B C G strain had to be used for guinea pig vaccination in the immunization experLment, none of the Iq-eeze-dried passages were acLm~stered in the ck~¢ form b u t were seeded and only the third to s ~ t h p ~ a g e was employed for liquid-vaccine production and all other investigations. Seeding on Souton~spototo " ~ 0
Singles possoges on Souton's medium
Freeze- drying
j
_
Fig. 1. Arr~'ageme~t o f m o d e l ~ e r ; . m e n t .
Assay of BCG strain immunimhg p o ~ ~ e in.aramizing potency of a strain was estimated on g-ain~ pigs in t e ~
of spleen weight. I t was agsumed that a guinea pig group solely imnaunized but not challenged represented a group given a vaccine of 100% efficacy. On the other'hand, a group solely infected but not d represented a group given an ineffective vaccine, i.e. one of 0% efficacy. Groups of guinea pigs were irranunized with the vaccines u n d e r study and after 1 month were challenged -with ~ I e n t erium tuberculo.~is, strain H 3 7 R v . Six wealds later the animals were killed and their spleens weighed. Mean spleen weights in the individual groups were evaluated statistically (Predc_hov~ & Hejdov~, 1971 ; Prbchov~i & gfr, 1972).
T o ~ lipids were determined via 48-h extraction of washed d ~ bacterial mass ~ t h hot 96% ethanol L'a a Soxhlet apparatus (hq~ra, Galliov~ & Hejdov~, 1971). T h e lipid content was e x p r e ~ e d (a) as percentage of bacterial ~ weight and Co) as percentage of a standard rrfixed-batch sample of Czechoslovak B C G stredn 725 S H ext,racted in parallel Protein assay was performed by determining total mtrogen by the KjeIdahl method and multiplying the values by a factor of 6.25. Polysao:harides were determined by the orcinol method after 21-day e~,'action of dry batten'a! mass ~ t h cold 8~/g if'CA (M~ra et aL, 1971).
NIorphoIogT was examined e l e c t r o n ~ c r o s c o p i ~ l y and the percentage of intact ceils was related to damaged cells. All ~ p l ~ were prepared directly from culture excludLng mechanical homogenization and using two t e c h n i q u ~ in order to ensure at least par'dal verification of results, as follow: 321
] . P R O C H O V A , M . l~i~.RA, H . ~ I O H E L S I ~ . ~ , Z. ~ f R A N D J. G A L L I O V ~ . (a) T h e ultrathin section technique of Ryter, K e l l e n b e ~ r , Birch-Anderson & l'viaaloe (1958) as modified by Mohelsk~f & Prllc~ov~ (1972) was supplemented by a prepolymerization step in pure Vestopal with 1% of activator and 1% of initiator at 4-9 °C for 24 h. Sections were cut on a 1 L K B Ultratome, contrasted with a 0-5% water solution o £ u r a n y I acetate £or 5 man and then ~ t h lead dt.~ate for 2-5 rain ( R e ~ o l d s , 1963). (b) T h e technique of whole specLmens repeatedly washed after fLxation with a 1% solution of O s O 4 in acetate--veronal buffer ~ e r Michaelis (Ryter et aL, 1958) with s~ine mad distilled water. T h e specimens were then applied to a foil and shaded with gold palladium at an angle of 20 °. A PbJlips E M 300 electron microscope was employed: acceleration voltage 80 kV, direct m a g n i f i ~ f i o n × 3 3 000 for u l t , r a ~ sections, × 12 500 for whole specimens. T h e criteria of 'pathologic~ d ~ a g e ' proposed for mycobacteria by A r m s t r o n g & d ' ~ r c y H a r t (1971) were used to expres~s major deviations from n o ~ I bacterial stracture. RESULTS T h e immunizing potency of strain B C G 725.SH decreased ~ e r freeze-drying ~ d subsequently gradually returned to noL~ial values both if the strain was passaged after one freezc-dr-~m4g run only (Fig. 2) or had been freeze-dried repeatedly (Fig. 3). T h e ing potency o f non-freeze-dried B C G 725 S H remained p r a c t i ~ l y unchanged L-~ the course of the experh-nent. immunization pofency Lipids recoicufoted to standard Upids
Percentage of intact ce! Is
r2C
IOC 8C 60 40
0
F
LI
Lx
Lz
Fig. 2. T h e c o m p a r i s o n o f ~ t m i z a t J o n p o t e n c y , lipid c o n t ~ t a n d n u m ~ r o f intact ceils Ln P r a g u e B C G s t ~ c~tiv~ted on SH m~um b e f o r e a n d after free~e-drying.
I n the b i o c h e ~ respect, B C G 725 S H displayed a significant d e c r e e o f the total lipid content ~ e r freeze-drying (Fig. 2). L a t e r in the course o f the experiment the t o ~ Hpids returned to the origh'~al values, similarly as did the in'LmtLnizkng potency, Lrrespective of whether t h e Z ~ r ~ had been freeze-dried once o r u p to four times (Fig. 3). T o ~ proteins, as c a l ~ l a t e d from t o ~ nitrogen a n d t o ~ ~lysaccharidez, displayed i n s i g a ~ t changes in both e x p e r i m e n ~ variants. I n electronmicroscopic representation, the perc~,m~e of intact m y c o b a c t e ~ in a 7-day culture of strain B C G 725 S H rose s u b s t ~ t i ~ l y after the first freeze-drylng (Fig, 2). W h e n freeze-dried once and s u b s e q u e n d y passaged, the s t r ~ exhibited a steep initial
322
CHANGES
IN BCG
VACCINE
STRAINS
"l
I00 80
40
'°io
F
LI
LII
LEI
[
L~
Fig. 3. T h e c o m p a r i s o n o f i m n a t m i ~ t i o n p o t e n c y , lipid c o n t e n t a n d nuxnber o f Lntact ceils in P r a g u e B C G s ~ cultivated o n S H m e d i u m before a n d after r e p e a t e d lyophilizatlon, K e y as in Fig. 2.
rise and then a gradual decrease in this value to the original l e v e l V,rith repeatedly freeze-dried lots the decrease in the percentage o f intact cells v.'~q far steeper. N o n freeze-dried lots did n o t show any substantial variation in intact mycobacteria percentage hu the course o f the experhnent (Fig. 3). Strain B C G 725 SAS behaved quite differently u n d e r i d e n t i ~ expetqanental conditions. After freeze-drying the zLng poten~¢ increased b u t n o t s and the percentage of h'~tact e l l s decreased, this decrease being significant. T h e inverse proportion o f these two parameters was thus r e d e d in both strains (725 S H and 725 SAS), although their values were reversed. T o t a l Iipids, on the other hand, did not change in either o f the exper;~ne~utal variants (one or several freeze-drying runs). I f these tRree properties are compared in the Danish strain and the two Czechoslovak strains 725 S H and 725 SAS (both o f t h e m reoresent the Danish strain modified by different conditions of maintenance) the Danish strain has the highest immunizing potency and lipid content. T h e Czechoslovak S H strain (non-freeze-dried, Former) ~ s p l a y e d a l-~gher Lm_munizing poten~r and lower percentage of lipids thin: strains SAS (Foimer'). T h e s e two properties are thus inversely proportional. I n neither instance, however, does the potency reach the values of the Danish strain. I n both Czechoslovak strains the pe o f intact c e ~ invariably inversely proportional to immunizing potency (Fig. 4). After freeze-dr-fi_ng, the in-umunizing potency o f the Danish s t u n remains above that of both of the Czechoslovak sh-ains. I n c o n ~ t to n o n - f r e e ~ - d r i e d strains, however, it is fly lower in 725 SH than 725 SAS. T h e percentage of intact cells after freezedrying is lower in 725 S H than 725 SAS. T h i s renders the relation bety~'een the percentage of intact cel~, immunizing potency and total lipids in both Czechoslovak strains after freeze-dEcing directly proportional, in c~ntrast to the pre-freeze-drying situation where only the influence of different culture media comes into pl~¢. However, the relation between irrumu.JzLng potency and percentage o f intact cells was inversely proportional in both strains before and after freeze-drying ~ k e (Fig. 5).
DISCUSSION As regards the immunizing potency, the Danish strain u ~ d in this study concomitantly v~th the two Czechoslovak strains has K e n c ~ d among the topmost strains by Jgspersen (1971) Lu his extensive e x p e r L m e n ~ -work..Another reason for our employing 323
J. P R O C H O V A ,
1VI, M A R A ,
H. 1VIOHELSKA,
Z. ~fR AND
J. G A L L I O V f l .
100 S0 0 |00 50 0
0
F
• ' SL
F
SL
F
SL
Fig. 4. T h e influence of Iyopb/Iization o n the irnrnunization potency, lipid c o n t e n t a n d ntumber o f intact cells in Prague B C G strain cultivated on S A S or S H m e d i m a n d in D a n i s h BCG stY. K e y as in Fig. 2. (F, F o y e r ; S L , freeze-drled vaccine.)
150
100 50 0 150
I00 50
0
]Fig. 5. ~ e influence o f oaltivatlon m e d i u m o n t h e ion potency, lipid c o n t e n t a n d n t L m b ~ o f intact c~l~ in Pre~g~ae B e G strain ¢ u h i ~ t e d on S A S o r S H n - t e ~ m a n d in D a n i s h B e G str',~n ~ ) . K e y as in ]Fig. 2. this strain as a standard of c vv~q t h a t b o t h o f t h e C ~ c h o s l o v a k s t r a i n s w e r e d e r i v e d f r o m it. T h u s , a c t u a l l y , t h e y r e p r e s e n t e d t w o v a r i a n t s o f s t r ~ u B C G C o p e n h a g e n i n v e h i c h m o d i f i c a t i o n s h a v e d e v - e l o p e d a8 a r e s u l t o f t h e d i s t i n c t m a i n t e n a n c e c o n d i ~ o n s of ~ch. Strain BCG 725 SH has been d in Sauton's medium ~ein h y d r o l y s a t e , w h i ~ haLs a r i c h c o n t e n t o f d i f f e r e n t a m / n o a c i d s . S a u t o n ' s o r i g i n a l m e d i u m , o n t h e o t h e r h a n d , o n l y c o n t a i n s o n e a m i n o a c i d , asparagine. S t r a i n B C G 725 S A S w a s o a h i v a t e d u p o n S a u t o n ' s p o t a t o , i.e. a s c o m p a r e d ~ 4 t h s t r a i n B C G C o p e n h a g e n , t h e c u l ~ r e m e d i u m w a s o o m s i d e r a b l y enric~ned b y t h e b i o d a e r n i ~ c o m p o n e n t s o f t h e p o t a t o . I t is t h e r e f o r e p r e s u m e d t h a t t h e s e B C G s t r a i n s w e r e ~ l y influenced by the culture media, markedly richer than those in which they were ~ maintained. The immun;~zing poten~ of the BCG vaccines was measured in immuni~tion experim_ents i n w h i c h t h e d i f f e r e n c e b e t w e e n g u i n e a p i g g r o u p s i n ~ c t e d o n l y a n d g r o u p s 324
g~ h~
Plate 1. BCG 725 SH, age 7 days. Cavitation starting in the nuclear re#on.
Plate2. BCG 725 SH, age 7 days, 3reaks in plasma membr~e and cell wall. CytopI~m is being released into he environment.
Plate 3. BCG 727 SAS, age 7 days, Cavitation in the nuclear region and autolysi s.
Plate 4, BCG 725 SAS. age 7 days, Mesosomal appmatus of the cell in lamdlar form.
Plate 5. BCG 725 SAS. age 7 days. M~osomal apparatus of the cell in tubuhr form,
~ g
:;. i ?
Plate 6. BCG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane.
\\
~late 7.
¢
.2
.
L.
3CG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane. Same as in Fig, I1.
i
°
7
C H A N G E S IN BCG V A C C I N E S T R A I N S i m m u n i ~ d and challenged "was evaluated. T h r e e criteria were available for evaluatg-~g the significance o f intergroup differences =mean spleen weight, Feldman's index (quantitative evaluation o f visceral T B lesions) and m e a n survival time. Since a positive correlation had previously been found between mean spleen weight, FeIdman's index and m e a n su.~ival time, mean spleen weight alone was used for the evaluation. T h i s m e t h o d has been used routinely on a long-term basis in our laborator$. I n order to ensure comparability between individual immunization experiments (Pr,Schov~i et aL, 1971) Prt$chovh introduced evaluation of results in percentages. T h e e~sence of this approach is outlined in the Materials and M e t h o d s section. Since 1964 we have regularly been checking the g potency of the D ~ s h vaccine w h e n routinely testing the Cze~oslova~k vaccine and f o u n d p e r m a n e n t l y high L,mmunming values with m i n o r . Pr-~chov~i et aL (I971) arrived at estimates of 94% arid 83% immunizing potency for the Danish and C ~ c h o s l o v a k vaccines, respectively. Since 1967 ~ e DarAsh BCG s t r ~ n has been maintained by the seed lot method. A s i g n ~ c a n t difference was found between the summary results of immunization experiments p d before and since the introduction of seed lot for r a c i n e p r o d u c t i o n ~ the values before freeze-dry~mg were somewhat higher t h ~ were those after the adoption of this preservation technio2ae. However, it should also be borne in m i n d that prior to its first freeze-drying the Danish s ~ n was cultivated on bile potato ~ d this factor could have exerted some influence on the changes in the immunizing potency and c h e m i s ~ o f the swain. Our routine resqalts over an 8-year period when the lipid, protein and polysaceiharide contents of the Czechoslovak B C G s ~ a i n SH and the Danish B C G strain -sere investigated were published previously (M~ra, gir, Galliovfi & HynEica, 1972). T h e highest s t a b i l i ~ in the contents of these three c o m p o n e n ~ during maintenance in the usual m a n n e r has been displayed by the Da~nish strain. After freeze-drcing, the lipid content d e c r e e d in both the Danish and the Czechoslovak strain, the protein content increased ira the Danish and d e c r e ~ e d in the Czechoslovak strain and the p o l y s a c c h ~ d e content r e m ~ e d stable in both. F r e e . e - d r y i n g also led to a decrease in the content of mycolie acids and waxes C and D. T h i s physical treah~nent had no effect on any o f the o t h e r fatty acids, as h~Lq been demonstrated by ~ t h paper mad gas ehrom (M~ra, glr, Hejdov.~i eg Mfirowf, 1973; Mfira, Julfik & gir, 1974). It was also found in previous experiments, by means of a spedMly devised method o f mycolie acid preparation by thin-layer chrom that freeze-dr)qng changed the ratio of the two main B C G mycolic acids (alpha and beta). T h i s ratio varied between different B C G strains (Michalec, t~d~ra & A d ~ c o v ~ , 1975 ; rvifira et aL, 1974). W h e n a new glycerine batch was u.~d for preparing culture m e d i u m f o r s t ~ in B C G 725 SH in previous experiments, the lipid and protein content increased while the polysaecharide content was si~ifiea.-atly decreased. Presumably, different contents of trace elements in the two glycerine batches m i g h t have been resp~onsible. Pathologically damaged bacteria were appraised according to the criteria proposed by Armstrong & d'Arcy Hart (1971). T h e s e authors worked with monolayer cultaares of peritoneal macrophages infected with .~'. tuberculosis H37Rv, JVL bG~,2s strain and B C G Moreau. T h e multiplicity of infection was far below 1, i.e. only some of the macrophages were infected with single mycobacteria. T h e resul~ were read electronmicro~copically and in parallel, in terrra o f viable cell counts. According to the f i n d i n ~ (total n u m b e r of 325
J. P R O C H O V A , M. ~ R A , bacteria in morphologies), 'p d by
H. I~¢IOHELSKA, Z. ~ I R A N D J. G A L L I O V t .
age cult,.ire as related to live bacteria and comping_son of bacterial ally damaged' bacteria were n o t viable. T h e divergencies & d ' ~ - c y Hart as pathological damage are as follows:
(I) Gross cavitations havLng n o Hint'ring membr-~te and tly affecting the nuclear region o f the cell. ~ e s e cavitations were frequent in tZhe present experim e n t s (Plate 1). (2) n of cytoplasmic contents through breaks in the plasrmx m e m b r a n e (and obviously the cell wall), i.e. autolysis. T h i s defect was also c o m m o n in the present experiments (Plate 2). (3) Lami , often leading to the form_adon of myelin-like figures. T h e y affect the cell wall and the electron-transparent zone surrou_nding it (the outer envelope of the cell). T h e s e forms were never e n ~ u n t e r e d in the present work. (4) Combinations of the above defects. I n the present material the frequent combination o f defects (1) and (2) ;~as d (Plate 3). According to these criteria, counts o f intact, d or dubious cells were made, the last attribute applies to cases where a safe decision was impossible, in partjc-alar because the was outside the section plane. Intact bacterial cture was mainly evaluated after Avakjan, Kac & Pavlova (1972). I t ~ rather complicated, e~speciaIly on account o f mesosomal apparatus polym o r p h y (Plates 4 and 5) and the presence of vacuoles, frequently of v e ~ bizarre shape, enveloped by a simple m e m b r a n e (Plates 6 and 7). T h e s e are considered b y Avakjmn to be e m p t y spaces after lipid L-~clusions, possibly extracted during specimen preparation. Although no standpoint is being taken here about the nat~are of these forms, their copious presence was indeed observed m oaltures of the B C G variants u n d e r study. T h e results obtained i n d i c t e d that, especially as far as strain B C G 72S BH ~ concerned, the Lmmunizing potency o f mycobacteria was directly proportional to the percentage o f intact cells. Hence the strain seems to contain two variants o f bacteria of which the one more resistant to mechanical action possesses a lower, and the more f ~ g i l e one a higher, immunizing potency. A change in the external conditions, in the present ~ e the p h y s i ~ effect of freeze-drying, brings about a selectAon of the variant o f superior resistance and h-fferior g potency. T h i s selection is incomplete, because after further cultivation in the usual m e d i u m and repeated freeze-drying fhe suppressed variant regains its prevalent position. Attention to the danger of mutation arising and selection during the m~intenance of B C G sta-aLns and during frec~-dryi_ng for the seedlot system is drawn ~ t h m u c h e~mphasis by G u l d (1971). T h e present findings support his views. T h e results o b t ~ n e d -with the Danish and the two ovak B C G strains that substantial changes develop after physical action (freeze-drying) only in st~ains being m ~ t a i n e d o n rich culture media. T h e t w o - m u t a n t t h e o ~ explains the dkfficulties encountered in t h e freeze-drylng of strain 725 SH. Although the decision was taken to use the most sparing m a n n e r ofdr)-mg, with all conditions adjusted accordingly (pre-freezing medium, etc.), the F~ of sur~¢ival was invariably and inva~iably the immunizing potency decreased. T h e present sta~dy was concerned with strains whose properties had been influenced by chemical (maintena~nce, m e d i u m m o ~ c a t i o n s ) and physical action (freeze-drying). T h e results .'o~licate that a straLn exposed to a certain kind o f influence (e.g. ~ e m i c a l ) 326
CHANGES
IN B e G
VACCINE STRAINS
reacts to action o f ~ o t h e r type (e.g. physical) i n a different way t h a n does a strain that has n o t b e e n acted u p o n i n t h e f o r m e r (chemical) m ~ e r , a n d v/ce versa. REFERENCES _Armstrong, J. A. & D,Arcy Hart, P. (1971). Response o f cultured macropkages to d4dycobacterium tuberculosis w i t h observafiorm o n fiasion of lysosomes ~ t h pEagosomes, ffou~,a/ of E x p e r l m ~ t a l 1 ~ , 713-740. Avakjan, A. A., Kac, L. ~q[., ~ Pavlova, I. B. (1972). Atlas aruztomii bakterij patogeun~ch alia geloveka i . rvledicina . G ~ d J. (1971). International S y m p o s i m o n B C G Vaccine, F r a ~ f i a r t m 1VIaLu, 1970. Sy~nposium Series in Immunobiological Standardisation, voL 17, pp. 143-146. Basel: Kargen. Jespersen, A. (1971). The Potenoj o f BCG- Vacdnes Determined on Animal's, p. 91. Cop : Statens S e r u m Instieate. l'dfira, 1H., Ga!!iov~, J. & Hejlov~, E. (1971). Wfiv kultiva~nich podmLuek n a chemick6 slo~eni pra~sktho a kodn-eisk~ho B C G ~ e _ n e . Studia Pn~motogica et P h t 31, 5/6, 196-206. rdfira, 1VI., J ~ , J. & ~ir, Z. (1974). Plynovfi chromatografie mastm#ch kyselin ~ z n # c h ~ e n _ f i B C G v r~azn3~cla kaltiva~_ic~h a l y o ~ z a ~ n i c h podminkfich. Studia Pneumologica et Ph ~ , 308-313. M~ra, M., ~ k , g . , Gnlliovfi, J. & Hyn~ica, V. (1972). Probl6my stabilit~y protituberkttl6zni v a k c ~ y . AHE,~/I 4, 100-1 ~ . M~ra, i . , ~ir, Z., Hejdov~, E. & ~t~fro-ca, E. (1973). St~_~diurnm ~ n n~kter#ch lipidick#ch frakci B e G , l ~ e n f a v kultivaEaxich a lyofi3iza~aaieh podmink~ch. Studia Pneumologica et Phtiseolo~ca ~ , 3/4, 254-263. Mie.halec, ~., Mfira, ?eL & Adamcov~i, D. (I975). Quantitative a n d q u ~ t a t i v e thin-layer c h r o m a t o g r a p h y o f mycolic acids in rium tuberculosis vat. bovis-BCG. Journal of Chromatography 104, 4 6 0 4 6 4 . 1VIohels~, H . & Pr~daov~, J. (1972). Comparative s t u d y o f fine m o r p h o l o g y o f t h e Czechoslovak a n d D a n i s h B C G vaccine strain. Journal of Hygiene, Epidemiology, Microbiology and Immunology 16, 328-333. Pnhchov§, J. & Hejdov~, E. (1971). Evaluation o f the protective effect o f t h e Prague B C G strain 725 Lu comparison with t h e D a n i s h strain 1331 (spleen weight h-a ~ainea pigs). m Series o f immuno Standards 17, 251-258. PrfiChov~, J. & ~ir, Z. (1972). C o m p m t i v e assay o f Lmrnunization potencies o f the C z e c h o slovak, D a n i s h a n d Japanese vaccines as a p r e l i w d n ~ step to t h e seed lot system in t h e p r o d u c t i o n o f B C G vaccine in Czechoslovakia. Progress in Immun Standardization S, 442 a,~6. Basel: Karger. Reynolds, E. S. (1963). T h e use o f lead citrate at h i g h p H as an d e c t r o n o p a q u e stain in d e c t r o n m i c r o s ~ p y , ffournal of Cell Biology 17, 208-211. Ryter, A., Kellenberger, E., Birch-_Andea-son, A. & NIaaloe, O. (1958) ~t~dde all microscope 6 h c t r o n i q u e de plasmas c o n t e n ~ t d e l ' a d d e d t s o ~ i b o n u e l t i q u e . I. Lea n u c l t o i d e s des bach&ties en crois~smuce active. Zeitschrift fiTr ZV.aturforschung 13b, 567-605.
327
S o m e bi morphol co elatio
cal, biochemic and al ch ges and their in selected B C G vaccine
.7. Pr:tchov~,t .~. 2d~ra,$ H . ~/loheIsk~,§ Z. ~gr§ and ff. Galliovd§
Changes m the biolo~caI, bloehem_i~ and morphological properties of three BeG s~ns, C o p e n h a g e n B C G 1331 a n d ~e¢o o f its d ~ v a t l v e s , P r a b ~ B C G 725 S H a n d P r a h a B C G 725 S A S , w e r e s t u d i e d a n d . T h e s e c h a n g e s w e r e c a u s e d b y c h e m i c a l (cult~.Ire medga) and p h y s i ~ factors (fre~z~-drying). T h e highest i.m_rnvJaizingl~t~ncy ~'~d the Im'ghest lipid content were displayed, both prior to and after ~reeze ch-ying, by the Da~_ish s~. cultivation of the Danish str-,dnon different media fin Prague resulted in a d e c r e a s e o f its p o t e n c y a n d lipid c o n t e n t . B o t h o f t h e C z e c h o s l o v a k s t r a i n s d e r i v e d in this m a n n e r , B C G 725 S H a n d B C G 725 S A S , d i f f e r e d m u t u a l l y b y t h e / r L m m t m l z i n g p o t e n c y , llpid c o n t e n t a n d o f L n t a ~ c~LLs. T h e i r r ' ~ a n i z i n g p o t e n c y a n d l i p i d c o n t e n t o f t h e s e t w o s t r a i n s w e r e m ~ v e r s e p r o p o r t i o n ; so w e r e t h e irnrnmnlzing potency and percenta~ of inta~ ria. The expe~mts s u g g e s t e d t h a t s u b s t a n t i a l c h a n g e s d e v e l o p a f t e r p h y s i c a l a c t i o n (freezed-~J;mg) only in stre~as being tx-~q_int~ed on f i ~ cultttre m e ~ F r ~ z e ~ r l n g changed the relationship betwedn Lng p o t e n ~ s-~d Hpld content into a direct one in both of the Czechoslovak straJ~ns. T~ne re~,ionship betweext ~ t m A z flag potenc~l and percentage of Lstact mycobactefia remained inverse. In B e G 72$ S H , total Hpids a n d L t r m a t m ~ g p o t e n c y a n d t h e percem-tag¢ o f k-~tact cells i n c r e a s e d a~er__~-eeze d r y i n g . I ~ B C G 725 S A S o n t h e o t h e r h a t t d t h e i r m x a u n ~ g poten~ fincreased, llpids z e m ~ e d o n a s t a b l e level a n d t h e o f L-tract cells dec~-~ased. T h e substant/al changes in t h e s e p induced by" Jfireeze-dt~Jng s u b s e q u e n t l y r e t u r n e d to t h e originaI v a l u e s i r r e s p e c t i v e o f w h e t h e r t h e strai_jxs h a d u n d e r g o n e a n u m b e r o f freeze-dr~_.ng p r o c e d u r e s o r h a d b e e n p a s s a g e d d i r e c t l y a f t e r a sfingle freeze-d-_~'ing v a n . Aza essential effect w a s t h u s o n l y p r o d u c e d b y t h e first f r e - e z e - ~ g , a n d t h e e.h~sagea ap//ear n o t t o b e stable. * R e c e i v e d f o r p u b l i c a t i o n 20 A p ~ 1976. f S t a t e I r ~ t i t u t e f o r D r u g C o n t r o l ( D i r . J; B~CL~ek, M . D . C . S c . ) , P r a g u e . ! s d H c r o b i o l o # M I n s t i t u t e , }J~_dlcal FacuIg¢, C h a r l e s U n i v e r s i W ( H e a d A s s i s t a n t P r o f e s s o r John, M.D.C.Sc.) Prague. § Institaate o f H y g i e n e a n d E p i (Dh-. P r o f e s s o r F . J a n d a , M . D . D . S c . ) , P r a g u e .
319
J . P R UoC H O
V
A, M . M A R A
H. I'vIOHELSKA, Z. ~IR A N D
J. G A L L I O V A
INTRODUCTION Although there has been a considerable decrease in t u ~ r c u l o s i s morbidity in Czechoslovakia, ha p a r t i c u l ~ among chiIdren and adolescents, prevention of a disease o f such consequence is still indispensable. T h e most i m p o r t a n t component of T B prevention is B C G vaccination. T h e original French B C G sti~in has, in the course of its long-term maintenance u n d e r varying cultivation conditions in different laboratories, undergone some substantial biological, b i o c h e m i ~ and m o r p h o l o ~ c a l changes. T h e selection of a variant of the strain and cultivation s that w ~ r a n t standard properties and t~igh vaccine effectiveness thus becomes a primary problem. Ac ~ e present steady was especially mined at investigating chmnges in the b i o l o g i ~ properties o f vaccine sh-ains of which the most important is Lrnmmaizing potency. AdditionaI aims were to explore, in a model experiment, w h e t h e r there were any concrete relations between the changes concerned (biological, biochemical and morphological) and to estimate the changes quantitatively. MATERIAL
AND
METHODS
Strains and cultivation Danish vaccine prepared from strain B C G 1331 was used as a standard. T h i s strain was pa~saged in Japan on bile-potato, then on asparagine-contaLaing original Sauton's m e d i u m and t h e n freeze-dried. On the basis of our l o n g - t e ~ experiences with this standard strain, the calculated percentage of immunizing potency o f the other s t r ~ s was related statistically 4o its Lrrm~unizing potency. T h e actual study was concerned with the Czechoslovak vaccine strah~ B C G 725, which h a d been derived from batch 725 of the Danish s ~ a i n in 1946. Since 1947 it has been passaged first on Sauton's potato and then d~ectly in Sauton's m e d i u m in which asparagine, has been replaced by e n ~ S c hydrolysate o f easeLn (SH medium). I n addition, the same B C G 725 strain was also studied after maintenance on Sauton's potato and cultivation in o r i ~ n a l Sauton's m e d i u m (SAS m e , u r n ) , containLng ~ p a r a g i n e as the only amino acid.
Freeze-drying of BCG-strab,.s Freeze-dr-ying was performed on an L Z 9B apparatus (Frigera, Kolin, Czechoslovakia). ~ e oalt~are m e d i u m of 72day ~ I t u r e s was remoo~d in a Birghaug b a ~ e r i a l press. T h e bacterial mass was homogenized by shaking with stainless steel balls and diI * U ted with 1~o Na.glutamate to a basic sus1~znsion of 50 m g o f ~ m i d r y wt./mL T h i s suspension was further diluted with 1 % Na-glutmmate to 10 mg[m_l, d i s p e n ~ d in 0.5 ml p o ~ o n s into a m p u l e s and f r o w n in a deep freezer t o - - 4 0 °C for 3 h. T h e anapoules were then transferred into the freeze-drying apparat~as and freez~e-dried for 16-18 h. Using a semiautomatic Edwards appliance, ampoule necks were con~_ricted, and d ~ n g was completed on an Edwards secondary freeze-drier (16 h); ampoules were seaIed u n d e r vacuum.
A
t of model experiment
A B C G stnLLn that had n o t previously been f r e e ~ - d r i e d (Former) was (I) freeze-dried or ( 2 ) s u b j e c t e d to further p ~ a g i n g . Tile freeze-dried strain was seeded and again either (1) pa~aged or (2) freeze-dried r e p u t e d l y (Fig. 1). I n this way three lines were obtained: 320
CHANGES I N B C G
VACCINE STRAINS
(1) non-freeze-dried strain; (2) ~ n freeze-dried once; (3) strain f r e e ~ - d r i e d repeatedly. Since liquid vaccine prepared from non-freeze-dried B C G strain had to be used for guinea pig vaccination in the immunization experLment, none of the Iq-eeze-dried passages were acLm~stered in the ck~¢ form b u t were seeded and only the third to s ~ t h p ~ a g e was employed for liquid-vaccine production and all other investigations. Seeding on Souton~spototo " ~ 0
Singles possoges on Souton's medium
Freeze- drying
j
_
Fig. 1. Arr~'ageme~t o f m o d e l ~ e r ; . m e n t .
Assay of BCG strain immunimhg p o ~ ~ e in.aramizing potency of a strain was estimated on g-ain~ pigs in t e ~
of spleen weight. I t was agsumed that a guinea pig group solely imnaunized but not challenged represented a group given a vaccine of 100% efficacy. On the other'hand, a group solely infected but not d represented a group given an ineffective vaccine, i.e. one of 0% efficacy. Groups of guinea pigs were irranunized with the vaccines u n d e r study and after 1 month were challenged -with ~ I e n t erium tuberculo.~is, strain H 3 7 R v . Six wealds later the animals were killed and their spleens weighed. Mean spleen weights in the individual groups were evaluated statistically (Predc_hov~ & Hejdov~, 1971 ; Prbchov~i & gfr, 1972).
T o ~ lipids were determined via 48-h extraction of washed d ~ bacterial mass ~ t h hot 96% ethanol L'a a Soxhlet apparatus (hq~ra, Galliov~ & Hejdov~, 1971). T h e lipid content was e x p r e ~ e d (a) as percentage of bacterial ~ weight and Co) as percentage of a standard rrfixed-batch sample of Czechoslovak B C G stredn 725 S H ext,racted in parallel Protein assay was performed by determining total mtrogen by the KjeIdahl method and multiplying the values by a factor of 6.25. Polysao:harides were determined by the orcinol method after 21-day e~,'action of dry batten'a! mass ~ t h cold 8~/g if'CA (M~ra et aL, 1971).
NIorphoIogT was examined e l e c t r o n ~ c r o s c o p i ~ l y and the percentage of intact ceils was related to damaged cells. All ~ p l ~ were prepared directly from culture excludLng mechanical homogenization and using two t e c h n i q u ~ in order to ensure at least par'dal verification of results, as follow: 321
] . P R O C H O V A , M . l~i~.RA, H . ~ I O H E L S I ~ . ~ , Z. ~ f R A N D J. G A L L I O V ~ . (a) T h e ultrathin section technique of Ryter, K e l l e n b e ~ r , Birch-Anderson & l'viaaloe (1958) as modified by Mohelsk~f & Prllc~ov~ (1972) was supplemented by a prepolymerization step in pure Vestopal with 1% of activator and 1% of initiator at 4-9 °C for 24 h. Sections were cut on a 1 L K B Ultratome, contrasted with a 0-5% water solution o £ u r a n y I acetate £or 5 man and then ~ t h lead dt.~ate for 2-5 rain ( R e ~ o l d s , 1963). (b) T h e technique of whole specLmens repeatedly washed after fLxation with a 1% solution of O s O 4 in acetate--veronal buffer ~ e r Michaelis (Ryter et aL, 1958) with s~ine mad distilled water. T h e specimens were then applied to a foil and shaded with gold palladium at an angle of 20 °. A PbJlips E M 300 electron microscope was employed: acceleration voltage 80 kV, direct m a g n i f i ~ f i o n × 3 3 000 for u l t , r a ~ sections, × 12 500 for whole specimens. T h e criteria of 'pathologic~ d ~ a g e ' proposed for mycobacteria by A r m s t r o n g & d ' ~ r c y H a r t (1971) were used to expres~s major deviations from n o ~ I bacterial stracture. RESULTS T h e immunizing potency of strain B C G 725.SH decreased ~ e r freeze-drying ~ d subsequently gradually returned to noL~ial values both if the strain was passaged after one freezc-dr-~m4g run only (Fig. 2) or had been freeze-dried repeatedly (Fig. 3). T h e ing potency o f non-freeze-dried B C G 725 S H remained p r a c t i ~ l y unchanged L-~ the course of the experh-nent. immunization pofency Lipids recoicufoted to standard Upids
Percentage of intact ce! Is
r2C
IOC 8C 60 40
0
F
LI
Lx
Lz
Fig. 2. T h e c o m p a r i s o n o f ~ t m i z a t J o n p o t e n c y , lipid c o n t ~ t a n d n u m ~ r o f intact ceils Ln P r a g u e B C G s t ~ c~tiv~ted on SH m~um b e f o r e a n d after free~e-drying.
I n the b i o c h e ~ respect, B C G 725 S H displayed a significant d e c r e e o f the total lipid content ~ e r freeze-drying (Fig. 2). L a t e r in the course o f the experiment the t o ~ Hpids returned to the origh'~al values, similarly as did the in'LmtLnizkng potency, Lrrespective of whether t h e Z ~ r ~ had been freeze-dried once o r u p to four times (Fig. 3). T o ~ proteins, as c a l ~ l a t e d from t o ~ nitrogen a n d t o ~ ~lysaccharidez, displayed i n s i g a ~ t changes in both e x p e r i m e n ~ variants. I n electronmicroscopic representation, the perc~,m~e of intact m y c o b a c t e ~ in a 7-day culture of strain B C G 725 S H rose s u b s t ~ t i ~ l y after the first freeze-drylng (Fig, 2). W h e n freeze-dried once and s u b s e q u e n d y passaged, the s t r ~ exhibited a steep initial
322
CHANGES
IN BCG
VACCINE
STRAINS
"l
I00 80
40
'°io
F
LI
LII
LEI
[
L~
Fig. 3. T h e c o m p a r i s o n o f i m n a t m i ~ t i o n p o t e n c y , lipid c o n t e n t a n d nuxnber o f Lntact ceils in P r a g u e B C G s ~ cultivated o n S H m e d i u m before a n d after r e p e a t e d lyophilizatlon, K e y as in Fig. 2.
rise and then a gradual decrease in this value to the original l e v e l V,rith repeatedly freeze-dried lots the decrease in the percentage o f intact cells v.'~q far steeper. N o n freeze-dried lots did n o t show any substantial variation in intact mycobacteria percentage hu the course o f the experhnent (Fig. 3). Strain B C G 725 SAS behaved quite differently u n d e r i d e n t i ~ expetqanental conditions. After freeze-drying the zLng poten~¢ increased b u t n o t s and the percentage of h'~tact e l l s decreased, this decrease being significant. T h e inverse proportion o f these two parameters was thus r e d e d in both strains (725 S H and 725 SAS), although their values were reversed. T o t a l Iipids, on the other hand, did not change in either o f the exper;~ne~utal variants (one or several freeze-drying runs). I f these tRree properties are compared in the Danish strain and the two Czechoslovak strains 725 S H and 725 SAS (both o f t h e m reoresent the Danish strain modified by different conditions of maintenance) the Danish strain has the highest immunizing potency and lipid content. T h e Czechoslovak S H strain (non-freeze-dried, Former) ~ s p l a y e d a l-~gher Lm_munizing poten~r and lower percentage of lipids thin: strains SAS (Foimer'). T h e s e two properties are thus inversely proportional. I n neither instance, however, does the potency reach the values of the Danish strain. I n both Czechoslovak strains the pe o f intact c e ~ invariably inversely proportional to immunizing potency (Fig. 4). After freeze-dr-fi_ng, the in-umunizing potency o f the Danish s t u n remains above that of both of the Czechoslovak sh-ains. I n c o n ~ t to n o n - f r e e ~ - d r i e d strains, however, it is fly lower in 725 SH than 725 SAS. T h e percentage of intact cells after freezedrying is lower in 725 S H than 725 SAS. T h i s renders the relation bety~'een the percentage of intact cel~, immunizing potency and total lipids in both Czechoslovak strains after freeze-dEcing directly proportional, in c~ntrast to the pre-freeze-drying situation where only the influence of different culture media comes into pl~¢. However, the relation between irrumu.JzLng potency and percentage o f intact cells was inversely proportional in both strains before and after freeze-drying ~ k e (Fig. 5).
DISCUSSION As regards the immunizing potency, the Danish strain u ~ d in this study concomitantly v~th the two Czechoslovak strains has K e n c ~ d among the topmost strains by Jgspersen (1971) Lu his extensive e x p e r L m e n ~ -work..Another reason for our employing 323
J. P R O C H O V A ,
1VI, M A R A ,
H. 1VIOHELSKA,
Z. ~fR AND
J. G A L L I O V f l .
100 S0 0 |00 50 0
0
F
• ' SL
F
SL
F
SL
Fig. 4. T h e influence of Iyopb/Iization o n the irnrnunization potency, lipid c o n t e n t a n d ntumber o f intact cells in Prague B C G strain cultivated on S A S or S H m e d i m a n d in D a n i s h BCG stY. K e y as in Fig. 2. (F, F o y e r ; S L , freeze-drled vaccine.)
150
100 50 0 150
I00 50
0
]Fig. 5. ~ e influence o f oaltivatlon m e d i u m o n t h e ion potency, lipid c o n t e n t a n d n t L m b ~ o f intact c~l~ in Pre~g~ae B e G strain ¢ u h i ~ t e d on S A S o r S H n - t e ~ m a n d in D a n i s h B e G str',~n ~ ) . K e y as in ]Fig. 2. this strain as a standard of c vv~q t h a t b o t h o f t h e C ~ c h o s l o v a k s t r a i n s w e r e d e r i v e d f r o m it. T h u s , a c t u a l l y , t h e y r e p r e s e n t e d t w o v a r i a n t s o f s t r ~ u B C G C o p e n h a g e n i n v e h i c h m o d i f i c a t i o n s h a v e d e v - e l o p e d a8 a r e s u l t o f t h e d i s t i n c t m a i n t e n a n c e c o n d i ~ o n s of ~ch. Strain BCG 725 SH has been d in Sauton's medium ~ein h y d r o l y s a t e , w h i ~ haLs a r i c h c o n t e n t o f d i f f e r e n t a m / n o a c i d s . S a u t o n ' s o r i g i n a l m e d i u m , o n t h e o t h e r h a n d , o n l y c o n t a i n s o n e a m i n o a c i d , asparagine. S t r a i n B C G 725 S A S w a s o a h i v a t e d u p o n S a u t o n ' s p o t a t o , i.e. a s c o m p a r e d ~ 4 t h s t r a i n B C G C o p e n h a g e n , t h e c u l ~ r e m e d i u m w a s o o m s i d e r a b l y enric~ned b y t h e b i o d a e r n i ~ c o m p o n e n t s o f t h e p o t a t o . I t is t h e r e f o r e p r e s u m e d t h a t t h e s e B C G s t r a i n s w e r e ~ l y influenced by the culture media, markedly richer than those in which they were ~ maintained. The immun;~zing poten~ of the BCG vaccines was measured in immuni~tion experim_ents i n w h i c h t h e d i f f e r e n c e b e t w e e n g u i n e a p i g g r o u p s i n ~ c t e d o n l y a n d g r o u p s 324
g~ h~
Plate 1. BCG 725 SH, age 7 days. Cavitation starting in the nuclear re#on.
Plate2. BCG 725 SH, age 7 days, 3reaks in plasma membr~e and cell wall. CytopI~m is being released into he environment.
Plate 3. BCG 727 SAS, age 7 days, Cavitation in the nuclear region and autolysi s.
Plate 4, BCG 725 SAS. age 7 days, Mesosomal appmatus of the cell in lamdlar form.
Plate 5. BCG 725 SAS. age 7 days. M~osomal apparatus of the cell in tubuhr form,
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Plate 6. BCG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane.
\\
~late 7.
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.2
.
L.
3CG 725 SAS, age 7 days. Vacuoles of unusual shapes surrounded by a simple membrane. Same as in Fig, I1.
i
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7
C H A N G E S IN BCG V A C C I N E S T R A I N S i m m u n i ~ d and challenged "was evaluated. T h r e e criteria were available for evaluatg-~g the significance o f intergroup differences =mean spleen weight, Feldman's index (quantitative evaluation o f visceral T B lesions) and m e a n survival time. Since a positive correlation had previously been found between mean spleen weight, FeIdman's index and m e a n su.~ival time, mean spleen weight alone was used for the evaluation. T h i s m e t h o d has been used routinely on a long-term basis in our laborator$. I n order to ensure comparability between individual immunization experiments (Pr,Schov~i et aL, 1971) Prt$chovh introduced evaluation of results in percentages. T h e e~sence of this approach is outlined in the Materials and M e t h o d s section. Since 1964 we have regularly been checking the g potency of the D ~ s h vaccine w h e n routinely testing the Cze~oslova~k vaccine and f o u n d p e r m a n e n t l y high L,mmunming values with m i n o r . Pr-~chov~i et aL (I971) arrived at estimates of 94% arid 83% immunizing potency for the Danish and C ~ c h o s l o v a k vaccines, respectively. Since 1967 ~ e DarAsh BCG s t r ~ n has been maintained by the seed lot method. A s i g n ~ c a n t difference was found between the summary results of immunization experiments p d before and since the introduction of seed lot for r a c i n e p r o d u c t i o n ~ the values before freeze-dry~mg were somewhat higher t h ~ were those after the adoption of this preservation technio2ae. However, it should also be borne in m i n d that prior to its first freeze-drying the Danish s ~ n was cultivated on bile potato ~ d this factor could have exerted some influence on the changes in the immunizing potency and c h e m i s ~ o f the swain. Our routine resqalts over an 8-year period when the lipid, protein and polysaceiharide contents of the Czechoslovak B C G s ~ a i n SH and the Danish B C G strain -sere investigated were published previously (M~ra, gir, Galliovfi & HynEica, 1972). T h e highest s t a b i l i ~ in the contents of these three c o m p o n e n ~ during maintenance in the usual m a n n e r has been displayed by the Da~nish strain. After freeze-drcing, the lipid content d e c r e e d in both the Danish and the Czechoslovak strain, the protein content increased ira the Danish and d e c r e ~ e d in the Czechoslovak strain and the p o l y s a c c h ~ d e content r e m ~ e d stable in both. F r e e . e - d r y i n g also led to a decrease in the content of mycolie acids and waxes C and D. T h i s physical treah~nent had no effect on any o f the o t h e r fatty acids, as h~Lq been demonstrated by ~ t h paper mad gas ehrom (M~ra, glr, Hejdov.~i eg Mfirowf, 1973; Mfira, Julfik & gir, 1974). It was also found in previous experiments, by means of a spedMly devised method o f mycolie acid preparation by thin-layer chrom that freeze-dr)qng changed the ratio of the two main B C G mycolic acids (alpha and beta). T h i s ratio varied between different B C G strains (Michalec, t~d~ra & A d ~ c o v ~ , 1975 ; rvifira et aL, 1974). W h e n a new glycerine batch was u.~d for preparing culture m e d i u m f o r s t ~ in B C G 725 SH in previous experiments, the lipid and protein content increased while the polysaecharide content was si~ifiea.-atly decreased. Presumably, different contents of trace elements in the two glycerine batches m i g h t have been resp~onsible. Pathologically damaged bacteria were appraised according to the criteria proposed by Armstrong & d'Arcy Hart (1971). T h e s e authors worked with monolayer cultaares of peritoneal macrophages infected with .~'. tuberculosis H37Rv, JVL bG~,2s strain and B C G Moreau. T h e multiplicity of infection was far below 1, i.e. only some of the macrophages were infected with single mycobacteria. T h e resul~ were read electronmicro~copically and in parallel, in terrra o f viable cell counts. According to the f i n d i n ~ (total n u m b e r of 325
J. P R O C H O V A , M. ~ R A , bacteria in morphologies), 'p d by
H. I~¢IOHELSKA, Z. ~ I R A N D J. G A L L I O V t .
age cult,.ire as related to live bacteria and comping_son of bacterial ally damaged' bacteria were n o t viable. T h e divergencies & d ' ~ - c y Hart as pathological damage are as follows:
(I) Gross cavitations havLng n o Hint'ring membr-~te and tly affecting the nuclear region o f the cell. ~ e s e cavitations were frequent in tZhe present experim e n t s (Plate 1). (2) n of cytoplasmic contents through breaks in the plasrmx m e m b r a n e (and obviously the cell wall), i.e. autolysis. T h i s defect was also c o m m o n in the present experiments (Plate 2). (3) Lami , often leading to the form_adon of myelin-like figures. T h e y affect the cell wall and the electron-transparent zone surrou_nding it (the outer envelope of the cell). T h e s e forms were never e n ~ u n t e r e d in the present work. (4) Combinations of the above defects. I n the present material the frequent combination o f defects (1) and (2) ;~as d (Plate 3). According to these criteria, counts o f intact, d or dubious cells were made, the last attribute applies to cases where a safe decision was impossible, in partjc-alar because the was outside the section plane. Intact bacterial cture was mainly evaluated after Avakjan, Kac & Pavlova (1972). I t ~ rather complicated, e~speciaIly on account o f mesosomal apparatus polym o r p h y (Plates 4 and 5) and the presence of vacuoles, frequently of v e ~ bizarre shape, enveloped by a simple m e m b r a n e (Plates 6 and 7). T h e s e are considered b y Avakjmn to be e m p t y spaces after lipid L-~clusions, possibly extracted during specimen preparation. Although no standpoint is being taken here about the nat~are of these forms, their copious presence was indeed observed m oaltures of the B C G variants u n d e r study. T h e results obtained i n d i c t e d that, especially as far as strain B C G 72S BH ~ concerned, the Lmmunizing potency o f mycobacteria was directly proportional to the percentage o f intact cells. Hence the strain seems to contain two variants o f bacteria of which the one more resistant to mechanical action possesses a lower, and the more f ~ g i l e one a higher, immunizing potency. A change in the external conditions, in the present ~ e the p h y s i ~ effect of freeze-drying, brings about a selectAon of the variant o f superior resistance and h-fferior g potency. T h i s selection is incomplete, because after further cultivation in the usual m e d i u m and repeated freeze-drying fhe suppressed variant regains its prevalent position. Attention to the danger of mutation arising and selection during the m~intenance of B C G sta-aLns and during frec~-dryi_ng for the seedlot system is drawn ~ t h m u c h e~mphasis by G u l d (1971). T h e present findings support his views. T h e results o b t ~ n e d -with the Danish and the two ovak B C G strains that substantial changes develop after physical action (freeze-drying) only in st~ains being m ~ t a i n e d o n rich culture media. T h e t w o - m u t a n t t h e o ~ explains the dkfficulties encountered in t h e freeze-drylng of strain 725 SH. Although the decision was taken to use the most sparing m a n n e r ofdr)-mg, with all conditions adjusted accordingly (pre-freezing medium, etc.), the F~ of sur~¢ival was invariably and inva~iably the immunizing potency decreased. T h e present sta~dy was concerned with strains whose properties had been influenced by chemical (maintena~nce, m e d i u m m o ~ c a t i o n s ) and physical action (freeze-drying). T h e results .'o~licate that a straLn exposed to a certain kind o f influence (e.g. ~ e m i c a l ) 326
CHANGES
IN B e G
VACCINE STRAINS
reacts to action o f ~ o t h e r type (e.g. physical) i n a different way t h a n does a strain that has n o t b e e n acted u p o n i n t h e f o r m e r (chemical) m ~ e r , a n d v/ce versa. REFERENCES _Armstrong, J. A. & D,Arcy Hart, P. (1971). Response o f cultured macropkages to d4dycobacterium tuberculosis w i t h observafiorm o n fiasion of lysosomes ~ t h pEagosomes, ffou~,a/ of E x p e r l m ~ t a l 1 ~ , 713-740. Avakjan, A. A., Kac, L. ~q[., ~ Pavlova, I. B. (1972). Atlas aruztomii bakterij patogeun~ch alia geloveka i . rvledicina . G ~ d J. (1971). International S y m p o s i m o n B C G Vaccine, F r a ~ f i a r t m 1VIaLu, 1970. Sy~nposium Series in Immunobiological Standardisation, voL 17, pp. 143-146. Basel: Kargen. Jespersen, A. (1971). The Potenoj o f BCG- Vacdnes Determined on Animal's, p. 91. Cop : Statens S e r u m Instieate. l'dfira, 1H., Ga!!iov~, J. & Hejlov~, E. (1971). Wfiv kultiva~nich podmLuek n a chemick6 slo~eni pra~sktho a kodn-eisk~ho B C G ~ e _ n e . Studia Pn~motogica et P h t 31, 5/6, 196-206. rdfira, 1VI., J ~ , J. & ~ir, Z. (1974). Plynovfi chromatografie mastm#ch kyselin ~ z n # c h ~ e n _ f i B C G v r~azn3~cla kaltiva~_ic~h a l y o ~ z a ~ n i c h podminkfich. Studia Pneumologica et Ph ~ , 308-313. M~ra, M., ~ k , g . , Gnlliovfi, J. & Hyn~ica, V. (1972). Probl6my stabilit~y protituberkttl6zni v a k c ~ y . AHE,~/I 4, 100-1 ~ . M~ra, i . , ~ir, Z., Hejdov~, E. & ~t~fro-ca, E. (1973). St~_~diurnm ~ n n~kter#ch lipidick#ch frakci B e G , l ~ e n f a v kultivaEaxich a lyofi3iza~aaieh podmink~ch. Studia Pneumologica et Phtiseolo~ca ~ , 3/4, 254-263. Mie.halec, ~., Mfira, ?eL & Adamcov~i, D. (I975). Quantitative a n d q u ~ t a t i v e thin-layer c h r o m a t o g r a p h y o f mycolic acids in rium tuberculosis vat. bovis-BCG. Journal of Chromatography 104, 4 6 0 4 6 4 . 1VIohels~, H . & Pr~daov~, J. (1972). Comparative s t u d y o f fine m o r p h o l o g y o f t h e Czechoslovak a n d D a n i s h B C G vaccine strain. Journal of Hygiene, Epidemiology, Microbiology and Immunology 16, 328-333. Pnhchov§, J. & Hejdov~, E. (1971). Evaluation o f the protective effect o f t h e Prague B C G strain 725 Lu comparison with t h e D a n i s h strain 1331 (spleen weight h-a ~ainea pigs). m Series o f immuno Standards 17, 251-258. PrfiChov~, J. & ~ir, Z. (1972). C o m p m t i v e assay o f Lmrnunization potencies o f the C z e c h o slovak, D a n i s h a n d Japanese vaccines as a p r e l i w d n ~ step to t h e seed lot system in t h e p r o d u c t i o n o f B C G vaccine in Czechoslovakia. Progress in Immun Standardization S, 442 a,~6. Basel: Karger. Reynolds, E. S. (1963). T h e use o f lead citrate at h i g h p H as an d e c t r o n o p a q u e stain in d e c t r o n m i c r o s ~ p y , ffournal of Cell Biology 17, 208-211. Ryter, A., Kellenberger, E., Birch-_Andea-son, A. & NIaaloe, O. (1958) ~t~dde all microscope 6 h c t r o n i q u e de plasmas c o n t e n ~ t d e l ' a d d e d t s o ~ i b o n u e l t i q u e . I. Lea n u c l t o i d e s des bach&ties en crois~smuce active. Zeitschrift fiTr ZV.aturforschung 13b, 567-605.
327