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Comparison of two types of freeze-dried BCG vaccine products
Journal
of Biological
Standardization
1974 2, 159-l 68
Comparison of two types of freeze-dried BCG vaccine products I. Experiments in the laboratory*
,7: L. Sirks,? L. Smith,t H. Cohen?
V. M. Sekhuis f and
Three lots of a freeze-dried BCG vaccine (P) prepared in the Netherlands from deep cultures of strain 1173 from Paris in glycerol-free medium were compared with four lots of a similar BCG vaccine (K) prepared in Copenhagen from surface-grown culttires of strain 1331. The three P lots induced larger local reactions in the skin of guinea-pigs and multiplied to a larger extent in spleens of mice than the four Danish lots, although the latter had a larger number of culturable particles per human dose. This confirms our previous experience and the reports of other investigators that the viable count is not a
sufficient criterion for the comparison of the quality of different BCG vaccines. Animal tests are indispensable for quality control. There existed a good correlation between both types of animal experiments. In a subsequent paper it will be shown that these results also correlated findings in man.
well with the
INTRODUCTION An investigation was undertaken to compare two differently prepared freeze-dried BCG vaccine products with respect to their reactivity in laboratory animals and in man. One vaccine was prepared in the Netherlands at the Rijks Instituut voor de Volksgezondheid (RIV) with bacteria grown in deep culture of the primary seed-lot 1173-P2 obtained from the Institut Pasteur in Paris in 1964. The other vaccine was prepared in Denmark at the Statens Seruminstitut with surface-grown bacteria of the Danish primary seed-lot 1331. The present paper reports the results of the laboratory tests of several lots of each product. The number of culturable particles per ml, the skin reactivity of guinea-pigs * Received for publication 7 February 1974. t Rijks Instituut voor de Volksgezondheid, Bilthoven, II
the Netherlands.
159
J. L. SIRKS
ET
AL.
and the maximum multiplication of the BCG bacteria in the spleens of mice were determined. The results of the animal tests showed that the Danish vaccine was less reactogenic than the Dutch vaccine. The results of the same vaccines in man will be given in a following paper in this journal (Sekhuis, Bleiker, Sirks & Cohen, 1974). MATERIALS
AND
METHODS
Production of the freeze-dried
vaccines
The production of both types of vaccine was based on the seed-lot system as prescribed in the International Requirements for dried BCG vaccine (W.H.O. Expert Committee on Biological Standardization, 1966). Three lots of the Netherlands vaccine (nos. P17, P18, P19) were prepared from the RIV primary seed-lot 1173-P3. The latter was prepared in our Institute in 1964 from a culture of the Paris seed-lot 1173-P2. Dr F. M. Levy (personal communication) tested seed-lot 1173-P3 in 1965 and found that it protected mice against tuberculous infection. The method of preparation of the Netherlands vaccine has been described previously (Sirks, Cohen & Smith, 1971). Briefly, the bacteria were grown in deep culture in essentially the same way as described by Ungar, Muggleton, Dudley & Griffiths (1962), except that 125 mg of magnesium sulphate/litre was added to the medium for the improvement of growth. The cells were harvested by centrifugation and resuspended in a new type of fluid carrier for freeze-drying which contained 8*3o/o Haemaccel R, 5% glucose and 0.025% Triton WR 1339. Haemaccel (Behringwerke AG, Marburg/Lahn), a derivative of gelatin with a molecular weight of about 35 000 (Schmidt-ThomC, Mayer & Schone, 1962), is not antigenic and is used as a plasma substitute in man (Schwick & Freund, 1962). The vaccine formed a porous cake which could be reconstituted very easily by the addition of water. The contents of the ampoules of some of the earlier production lots had a glassy surface which made reconstitution difficult. This was improved by raising the sublimation temperature in the ampoules at an earlier stage in the drying process. Although the percentage of survival of the bacteria was somewhat lower, reconstitution was much easier. The survival of the bacteria as determined by counting the number of culturable particles before and after freeze-drying remained within acceptable limits. The residual moisture content of the product was between 0.5 and 1%. Four lots of Danish freeze-dried BCG vaccine (nos. K32, K41, K46, K48) were kindly supplied by Dr K. Bunch-Christensen of the Statens Seruminstitut. They were prepared by a uniform technique according to the routine method used in Copenhagen. Briefly, cultures from primary seed-lot 1331 were grown on the surface of Sauton medium. The collected bacterial mass was ground in a ball mill and resuspended before freeze-drying in a 1.5% sodium glutamate solution. All lots of each type of vaccine product meet the international requirements for potency and safety of dried BCG vaccine (W.H.O. Expert Committee on Biological Standardization, 1966). The ampoules of all vaccines were stored at -20 “C. Determination
of number of culturable particles
The number of culturable particles was determined by inoculating different dilutions of the reconstituted vaccines on Lowenstein-Jensen medium and counting the number of colonies that developed after one month of incubation at 37 “C. 160
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The content of each ampoule of lot P19 of the Netherlands vaccine was reconstituted with O-2ml of saline and the content of each ampoule of the lots P17 and P18 with 2-Oml. These volumes were the same as were used to fill the vaccine into the ampoules. The reconstitution volumes per ampoule for human administration, however, were O-5 and 5 ml, respectively. The content of each ampoule of the Danish vaccine was reconstituted with 5 ml of saline as recommended for human vaccination. The counts for each product were expressed as the number of culturable particles per human dose (0.1 ml). Guinea-pig skin reactivity test Each ampoule of vaccine was reconstituted as recommended for human use for the test of skin reactivity of guinea-pigs (W.H.O. Requirements section 5.3.2.). We used a modification of the method introduced by Jensen (1946). Details of the method used have been published elsewhere (Sirks & Cohen, 1972). The vaccines were tested in pairs; each pair on eight guinea-pigs. This was done by injecting two lots on the same guinea-pig in order to make a direct comparison of the reactions and to avoid differences in the response between guinea-pigs (Pierce, Dubos & Schaefer, 1956). Two doses of each lot were used instead of the usual four to reduce to a minimum the influence of injection reactions on each other. The doses were 0.1 ml of the reconstituted undiluted vaccine and 0.1 ml of a 1 : 10 dilution. The three Netherlands lots, P17, P18 and P19, were paired with each other and each of the four Danish lots were paired with lot P17. The latter served as a reference for comparison between the Danish lots and between the Danish and the Netherlands lots. The diameters of the skin-reactions were measured twice a week during six weeks. The maximum responses developed around three weeks (see Fig. 1). The size of the maximum reactions provided better data for analysis of the strength of the vaccines than the time of duration of the reactions (Jensen, 1946), which is difficult to determine. 8
3
7
IO
14
17
21 24
28
31
35
38
42
Time (days)
Fig. 1. The development of the skin reactions on guinea-pigs during 42 days following inoculation of two doses of lot P17 (Netherlands) and of lot K41 (Danish). Each point represents the mean of the diameters of the reactions on eight guinea-pigs.
161
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ET
AL.
Mouse virulence test
In addition to the Jensen-test, the determination of the virulence of each vaccine was based also on the multiplication of the BCG organisms in the spleens of mice (Pierce, Dubos & Schaefer, 1953, 1956; L&y, Mande, Conge, Fillastre & Orssaud, 1968). Details of our method were published earlier (Sirks, 1973). On three consecutive days the content of one ampoule of each of the seven lots of vaccine was reconstituted and diluted in O.lo/o bovine albumin solution in such a way that the volume of O-2ml to be injected into each mouse contained one hundredth of a human dose. Each preparation was injected intravenously into three 30-35-day-old female mice of the RIV Swiss strain. The test was repeated on three consecutive days one month later. In this way vaccine from six ampoules of each lot was injected into six groups of three mice each. The optimum time to examine the spleens was determined in a series of preliminary experiments with the same lots in which mice were killed after one, three and six weeks. It was then found that the number of the BCG bacteria in the spleens was maximal about three weeks after injection. Therefore, the mice were always killed three weeks after inoculation. Only two of the three mice injected with each lot were killed and the spleens removed. The third mouse in each group served as a replacement in case of an intercurrent death. The number of culturable BCG particles in each spleen was determined as follows: the spleen was ground in a Potter tube in 10 ml of 0.1% bovine albumin solution, then diluted 1 : 10 and 1 : 100; 0.2 ml of each dilution was inoculated in duplicate on Liiwenstein-Jensen medium. Colony counts were made after one month of incubation and the number of culturable particles recovered from each spleen was calculated.
RESULTS Number of culturable particles and stability Table 1 gives the results of the colony counts estimated per human dose of each lot of vaccine after storage at -20 “C and after being held for one month at 37 “C. The Netherlands vaccine had about one-half of the number of culturable particles per human dose as was present in the Danish vaccine. The results of the heat-stability test obtained with each product showed little variations. 1. Number of culturable particles per human dose for three lots of a Netherlands vaccine and four lots of a Danish vaccine after storage at - 20 “C and after one month at 37 “C
TABLE
Culturable particles in thousands Vaccine Netherlands
Lot no.
Storage at -20 “C
After 1 month 37 “c’
Survival (%)
P17
200 336 236
80 184
440 440
200 150 270 160
40 55 43 45 34 63 41
P18 P19
Danish
K32 K41 K46 K48
162
430 390
104
FREEZE-DRIED Guinea-pig
BCG
skin reactivity
VACCINES.
I
test
An example of the reactions of guinea-pigs to the inoculation of two amounts each of P17 and lot K41 is given in Fig. 1. The lesions induced by both vaccines reach their maximum diameter between 17 and 24 days after injection. Similar results were observed with the other lots of vaccine of each product. Table 2 gives the mean diameter of the lot
TABLE
2. Mean diameters of skin reactions three weeks after injection 0.1 ml Pairs of vaccine lots* Pl8 P17 PI8 -i P19 P17 { P19 P17 -C K48 P17 1 K46 P17 -C K41 P17 K32
Undiluted 8.2.f 7.4 8.9 8.4 7.7 8.2 7.5 6.2 7.4 6.7 7.0
5.1 9.6 8.8
of
1 : 10 8.0 6.4 7.7 6.7 5.2 5.9 5.7 4.4 6.5 5.6 5.9 3.6 8.6 7.8
* Each pair was tested on eight guinea-pigs. t Mean diameter (mm) of the eight reactions.
reactions induced by each test dose of each of the paired lots on eight guinea-pigs that were read at three weeks after injection. This time was optimum for evaluation, as shown in Fig. 1. A comparison of the reactivity of each pair of lots tested is shown in Fig. 2. Each point represents the difference between the mean diameter of the reactions induced by one lot on a single guinea-pig and the mean diameter of the reactions induced by the other lot tested on the same guinea-pig. The Netherlands vaccines were more reactogenic than the Danish vaccine. P18 appeared to be the most reactogenic. It induced larger reactions than P17 on seven of eight guinea-pigs and the reactions were larger than or equal to those caused by P19. On the other hand, P17 was more reactogenic than each of the four Danish vaccine-lots. Their comparison with P17 suggests that there was no difference between their reactivity on guinea-pigs. Mouse virulence test
Table 3 gives the calculated number of culturable particles of BCG bacteria per spleen. For each lot of vaccine the geometric mean of individual values appears in the last column. For statistical analysis individual values were transformed to a logarithmic scale. Then the mean value of the logarithm (to base 10) of the number of culturable particles for the two mice in each group was calculated. This provided a figure for each combination of one of the six injection days and each of the seven lots of vaccine. An analysis of variance was applied to these data. The variation between lots of vaccine was split into three parts, viz. the contrast between P and K vaccines, differences between 163
J. L.
SIRKS
ET AL.
the three lots of P vaccine and differences between the four lots of K vaccine. The results of the analysis are shown in Table 4.
.
5
f 4r 3 . !! 2-• .s I--A 2 Q B 5 -I0g -3--2- . PM-PI7
t.f,r: . as ..
t.
.8 .
. .
? .
0.. Pl6-PI9
Pl7-PI9
P17-K46
P17-K46
PI7-K4l
Pl7-K32
Fig. 2. Comparison of pairs of vaccine lots in groups of eight guinea-pigs. Differences on individual guinea-pigs between mean diamater of skin reactions to two doses of one vaccine lot and mean diameter of reactions to two doses of one other vaccine lot, three weeks after inoculation.
There were rather large differences between individual mice. Nevertheless the figures demonstrate clearly that many more culturable particles are found in the spleen three weeks after injection of one-hundredth of a human dose of the Netherlands (P) vaccine lots than three weeks after injection of the same dose of the Danish (K) vaccine lots. It is also evident that P18 is more virulent for mice than P17 and P19. No systematic differences were detected, neither between P17 and P19 nor among the four lots of K vaccine.
DISCUSSION The foregoing results show that it was possible to prepare a stable and potent freeze-dried vaccine, which meets the W.H.O. Requirements, by cultivation of the strain 1173-P2 in deep culture. Brindle, Griffiths, Holme, Stalker, Burland, Coates & Muggleton (1972) have shown earlier that this is also possible with seed-lot 1331 from Copenhagen and not merely with seed-lot 1077 which is normally used in England for the preparation of vaccine from deep cultures. In this investigation we wanted to compare two types of vaccines and not merely two strains. Therefore we used the vaccine suspensions after reconstruction as recommended for use in man as a starting point for further diluting them to the same extent for testing in animals. This is in contradistinction to the method of several investigators who based their dilution-scheme on equal viable counts or equal dry weights (e.g. Pierce et al., 1956; Benoit & Panisset, 1963 ; Frappier, Portelance, St. Pierre & Panisset, 1971; and others).
The results of our experiments demonstrate that the three vaccine lots prepared by us from the Paris strain all gave larger local vaccination reactions in guinea-pigs and multiplied to a larger extent in spleens of mice than the four vaccine lots prepared in Copenhagen from seed-lot 1331, although the latter had about twice the number of culturable particles per human dose. 164
II.3 32.3 28.2 * 0.24 I.4 0.51
12.5 97.2 29.7 0.27 * 0.46 0.76
20.3 31.3 18.5 * 0.87 0.71 1.6
Group 3 15.9 55.0 13.9 3.0 * 2.6 0.91
Ifs*0 41.6 80.6 9.0 3.5 I.2 2.8
18-l 140.2 107.3 044 4.2 2.0 * the calculated
11.1 91.8 72.9 0.95 12.5 * 0.23
particles in thousands per spleen Test 2 A , c Group 4 Group 5
of culturable
23.6 60.0 5.0 2.3 I.6 0.46 *
Estimated number Test 1 A Group 2
estimate
86.7 39.3 IO.0 6.1 0.23 2-o 4.7
,
16.2 64.6 27.5 1.20 0.96 1.18 0.68
Geometric meant for 12 mice
mice,
if one colony had grown
5.2 72.5 17.9 0.25 9.4 11-l 0.23
Group 6
of BCG in spleens of mice. Estimated number of cuhurable particles in the spleen of individual three weeks after intravenous injection of one-hundredth of a human dose of vaccine
10.2 124.8 36.4 7.0 o-43 I.3 2.3
Group 1
25.7 84.1 39.1 6.6 0.68 I.6 0.71
f
3. Multiplication
* No colonies developed on the media inoculated with the 1 : 100 or 1 : 10 dilutions. t For the counts of spleens from which no growth was obtained, we assigned a value of one-half from the 1 : 10 dilution.
PI7 P18 P19 K32 K41 K46 K48
Vaccine lot
TABLE
J. L. SIRKS
ET AL.
TABLE 4. Analysis of variance, applied to mean values calculated for each of 42 groups of two mice denoted in Table 3 (each mean value regards the logarithm of the estimated number of culturable particles in the spleens of two mice) Source of variation Between days Between vaccine lots -P-lots versus K-lots
Degrees of freedom 5 6 1 2 3
-Between P-lots -Between Residual Total
K-lots 30 41
Sum of squares
Mean square
1.161 24.356 23.006 l-114 0.236 4.244 29.760
0.232
1.64
N.S.
23.006 0.557 0.079 0.141
162.6 3.94 0.56
P
F-ratio
Significancy
These findings confirm the experience of other investigators that the viable count as such is not a sufficient criterion for the comparison of the quality of BCG vaccines of different origins (Levy et al., 1968; Frappier et al., 1971). The viable count, however, has value as a check on the degree of consistency in the production of consecutive lots of a vaccine prepared from the same strain in the same way and as a check on the stability of the vaccine during freeze-drying and during storage at different temperatures (W.H.O. Expert Committee on Biological Standardization, 1966). For the comparison of vaccines, prepared in different ways from different substrains, animal experiments are necessary for potency and safety evaluation. These tests are also of value in the control of routine production of successive lots of the same product. This was demonstrated by the fact that a slight but distinct difference in potency between the Netherlands lot P18 and the two other lots P17 and P19 could be found in this way. The Jensen-test is recommended in the W.H.O. Requirements for dried BCG vaccine (1966) under ‘Safety-tests’. It may, however, serve simultaneously as a potency test. If a vaccine induces larger reactions in guinea-pigs than a reference preparation one might expect that it would be able also to elicit a larger and longer lasting Mantoux-reactivity and possibly give better protection in man (Levy et al., 1968; Jespersen, 1971; Guld, 1971). On the other hand, if a vaccine induces significantly larger reactions than a reference preparation with an acceptable reactogenicity for man it might cause undesirable vaccination reactions. The test on the multiplication of the BCG bacteria in the spleen of mice is mainly a potency test. Dubos & Pierce (1956) concluded earlier that ‘there is no doubt that immunity is an expression of the ability of the BCG organisms to multiply in the body of the vaccinated individuals’. In general differences between two vaccines with respect to their rate of multiplication in low doses.
in the spleen of mice are observed only if they are injected
The best time for the determination of the number of culturable particles in the spleens is when multiplication is maximal. We found that for most vaccines this time was three weeks after intravenous injection. In this way we found for the P and K vaccines a good correlation between the results of the mouse potency test and the Jensen-test. The greater virulence of the P vaccines in these experiments may be due chiefly to the
substrain from which it has been prepared. Ladefoged, Bunch-Christensen (1970) found that a liquid
& Guld
vaccine made from seed-lot 1173-P2 gave better protection
in
low doses in bank voles against virulent tubercle bacilli tban a liquid vaccine which was 166
FREEZE-DRIED
BCG VACCINES.
I
prepared from the Danish seed-lot 1331 in exactly the same way. It cannot be excluded, however, that besides the properties of the strain the method of preparation has some influence on the potency of the vaccine. Homogenization of the bacterial mass by grinding in a ball mill is avoided by application of the deep-culture technique. This may result in a reduced number of killed or damaged mycobacteria. The two types of vaccines we studied did not only differ in the substrain used, the method of cultivation and the method of harvesting and resuspending the bacteria but also in the type of fluid carrier for freezedrying. Any one of these factors alone or combined may have contributed to differences between the Netherlands and the Danish vaccines. None of these factors, however, can account for the fact that one of the three P vaccines was somewhat more virulent than the others. The results of the field trial, to be published in the second part of this communication (Sekhuis et al., 1974) show that the two types of vaccine induced in man post-vaccinal Mantoux- and vaccination reactions which were in agreement with their reactogenicity in guinea-pigs and their multiplication rate in mice as described in this paper. Acknowledgements We wish to thank Dr K. Bunch-Christensen, Statens Seruminstitut, for supplying the Danish vaccines; Dr F. M. L&y, International Children’s Centre Paris, for the testing of seed-lot 1173-P3 in mice; Dr Margaret Pittman, Washington D.C., U.S.A., for helpful comments and criticism of the manuscript; Mr R. W. Mendels, Mr P. van Putten and Mr P. Vonk for their technical assistance.
REFERENCES Benoit, J. L. & Panisset, Mi (1963). Survie et multiplication du BCG et du bacille tuberculeux chez la souris. IV. Acta Tuberculosea et Pneumologica Scandinavica 43, 125-136. Brindle, T. W., Griffiths, M. I., Holme, T., Stalker, R., Burland, W. L., Coates, G. A. & Muggleton, P. W. (1972). A trial to compare reactions and responses to BCG vaccine prepared from Copenhagen 1331 and Glaxo 1077 strains. Tubercle, London 53, 100-105. Dubos, R. J. & Pierce, C. H. (1956). Differential characteristics in vitro and in vivo of several substrains of BCG. IV. Immunizing effectiveness. American Review of Tuberculosis and Pulmonary Diseases 74, 699-717. Frappier, A., Portelance, V., St. Pierre, J. & Panisset, M. (1971). BCG-strains: characteristics and relative efficacy. In: Status of Immunization in Tuberculosis in 1971. Report of a conference on progress to date, future trends and research needs. DHEW Publication no. (NIH) 72-68, pp. 157-178. Guld, J. (1971). BCG as an immunizing agent. In: Status of Immunization in tuberculosis in 1971. Report of a conference on progress to date, future trends and research needs. DHEW Publication no. (NIH) 72-68, pp. 149-156. Jensen, K. A. (1946). Practice of the Calmette vaccination. Acta Tuberculosea Scandinavica 20, l-45. Jespersen, A. (1971). The potency of BCG-vaccines determined on animals. Thesis, Copenhagen, Chapter 5, pp. 82-83. Ladefoged, A., Bunch-Christensen, K. & Guld, J. (1970). The protective effect in bank voles of some strains of BCG. Bulletin of the World Health Organization 43, 71-90. L&y, F. M., Mande, R., Conge, G., Fillastre, C. & Orssaud, E. (1968). Perspectives for BCG standardization. Advances in Tuberculosis Research Vol. 16, Chapter IV-3, pp. 124-128. Cane& G., Birkhluser, H., & Block, H., eds. New York: Karger, Basel. Pierce, C. H., Dubos, R. J. & Schaefer, W. B. (1953). Multiplication and survival of tubercle bacilli in the organs of mice. Journal of Experimental Medicine 97, 189-206. 167
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Pierce, C. H., Dubos, R. J. & Schaefer, W. B. (1956). Differential characteristics in vitro and in wivo of several substrains of BCG. III. Multiplication and survival in viuo. American Review of Tuberculosis and Pulmonary Diseases 74, 683-698. Schmidt-Thorn& J., Mayer, A. & Schone, H. H. (1962). Zur Chemie eines neuen Plasmaexpanders. Arzneimittelforschung 12, 378-380. Schwick, G. & Freund, U. (1962). Immunologische Untersuchungen mit Haemaccel. Deutsche Medizinische Wochenschrift 87, 737-741. Sekhuis, V. M., Bleiker, M. A., Sirks, J. L. & Cohen, H. (1974). Comparison of two types of freeze-dried BCG vaccine products. II. Results of vaccination of men. Journal of Biological Standardization 2, 169-181. Sirks, J. L. (1973). Controle van BCG-vaccin door bepaling van de mate van vermenigvuldiging in muizen. Verslagen en Mededelingen betreffende de Volksgezondheid 26, 190-193. Sirks, J. L. & Cohen, H. (1972). Controle van BCG-vaccin volgens Jensen door intracutane injecties bij cavia’s. Verslagen en Mededelingen betreffende de Volksgezondhe-id 24, 206212. Sirks, J. L., Cohen, H. & Smith, L. (1971). Production of freeze-dried BCG-vaccine. Symposia Series in Immunobiological Standardization 17, 163-168. Ungar, J., Muggleton, P. W., DudIey, J. A. R. & Griffiths, M. I. (1962). Preparation and properties of a freeze-dried BCG-vaccine of increased stability. British MedicalJournal II, 1086-1089. W.H.O. Expert Committee on Biological Standardization (1966). Requirements for dried BCG-vaccine. W.H.O. Technical Report Series no. 329, Annex 1, pp. 25-48.
168
of Biological
Standardization
1974 2, 159-l 68
Comparison of two types of freeze-dried BCG vaccine products I. Experiments in the laboratory*
,7: L. Sirks,? L. Smith,t H. Cohen?
V. M. Sekhuis f and
Three lots of a freeze-dried BCG vaccine (P) prepared in the Netherlands from deep cultures of strain 1173 from Paris in glycerol-free medium were compared with four lots of a similar BCG vaccine (K) prepared in Copenhagen from surface-grown culttires of strain 1331. The three P lots induced larger local reactions in the skin of guinea-pigs and multiplied to a larger extent in spleens of mice than the four Danish lots, although the latter had a larger number of culturable particles per human dose. This confirms our previous experience and the reports of other investigators that the viable count is not a
sufficient criterion for the comparison of the quality of different BCG vaccines. Animal tests are indispensable for quality control. There existed a good correlation between both types of animal experiments. In a subsequent paper it will be shown that these results also correlated findings in man.
well with the
INTRODUCTION An investigation was undertaken to compare two differently prepared freeze-dried BCG vaccine products with respect to their reactivity in laboratory animals and in man. One vaccine was prepared in the Netherlands at the Rijks Instituut voor de Volksgezondheid (RIV) with bacteria grown in deep culture of the primary seed-lot 1173-P2 obtained from the Institut Pasteur in Paris in 1964. The other vaccine was prepared in Denmark at the Statens Seruminstitut with surface-grown bacteria of the Danish primary seed-lot 1331. The present paper reports the results of the laboratory tests of several lots of each product. The number of culturable particles per ml, the skin reactivity of guinea-pigs * Received for publication 7 February 1974. t Rijks Instituut voor de Volksgezondheid, Bilthoven, II
the Netherlands.
159
J. L. SIRKS
ET
AL.
and the maximum multiplication of the BCG bacteria in the spleens of mice were determined. The results of the animal tests showed that the Danish vaccine was less reactogenic than the Dutch vaccine. The results of the same vaccines in man will be given in a following paper in this journal (Sekhuis, Bleiker, Sirks & Cohen, 1974). MATERIALS
AND
METHODS
Production of the freeze-dried
vaccines
The production of both types of vaccine was based on the seed-lot system as prescribed in the International Requirements for dried BCG vaccine (W.H.O. Expert Committee on Biological Standardization, 1966). Three lots of the Netherlands vaccine (nos. P17, P18, P19) were prepared from the RIV primary seed-lot 1173-P3. The latter was prepared in our Institute in 1964 from a culture of the Paris seed-lot 1173-P2. Dr F. M. Levy (personal communication) tested seed-lot 1173-P3 in 1965 and found that it protected mice against tuberculous infection. The method of preparation of the Netherlands vaccine has been described previously (Sirks, Cohen & Smith, 1971). Briefly, the bacteria were grown in deep culture in essentially the same way as described by Ungar, Muggleton, Dudley & Griffiths (1962), except that 125 mg of magnesium sulphate/litre was added to the medium for the improvement of growth. The cells were harvested by centrifugation and resuspended in a new type of fluid carrier for freeze-drying which contained 8*3o/o Haemaccel R, 5% glucose and 0.025% Triton WR 1339. Haemaccel (Behringwerke AG, Marburg/Lahn), a derivative of gelatin with a molecular weight of about 35 000 (Schmidt-ThomC, Mayer & Schone, 1962), is not antigenic and is used as a plasma substitute in man (Schwick & Freund, 1962). The vaccine formed a porous cake which could be reconstituted very easily by the addition of water. The contents of the ampoules of some of the earlier production lots had a glassy surface which made reconstitution difficult. This was improved by raising the sublimation temperature in the ampoules at an earlier stage in the drying process. Although the percentage of survival of the bacteria was somewhat lower, reconstitution was much easier. The survival of the bacteria as determined by counting the number of culturable particles before and after freeze-drying remained within acceptable limits. The residual moisture content of the product was between 0.5 and 1%. Four lots of Danish freeze-dried BCG vaccine (nos. K32, K41, K46, K48) were kindly supplied by Dr K. Bunch-Christensen of the Statens Seruminstitut. They were prepared by a uniform technique according to the routine method used in Copenhagen. Briefly, cultures from primary seed-lot 1331 were grown on the surface of Sauton medium. The collected bacterial mass was ground in a ball mill and resuspended before freeze-drying in a 1.5% sodium glutamate solution. All lots of each type of vaccine product meet the international requirements for potency and safety of dried BCG vaccine (W.H.O. Expert Committee on Biological Standardization, 1966). The ampoules of all vaccines were stored at -20 “C. Determination
of number of culturable particles
The number of culturable particles was determined by inoculating different dilutions of the reconstituted vaccines on Lowenstein-Jensen medium and counting the number of colonies that developed after one month of incubation at 37 “C. 160
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VACCINES.
I
The content of each ampoule of lot P19 of the Netherlands vaccine was reconstituted with O-2ml of saline and the content of each ampoule of the lots P17 and P18 with 2-Oml. These volumes were the same as were used to fill the vaccine into the ampoules. The reconstitution volumes per ampoule for human administration, however, were O-5 and 5 ml, respectively. The content of each ampoule of the Danish vaccine was reconstituted with 5 ml of saline as recommended for human vaccination. The counts for each product were expressed as the number of culturable particles per human dose (0.1 ml). Guinea-pig skin reactivity test Each ampoule of vaccine was reconstituted as recommended for human use for the test of skin reactivity of guinea-pigs (W.H.O. Requirements section 5.3.2.). We used a modification of the method introduced by Jensen (1946). Details of the method used have been published elsewhere (Sirks & Cohen, 1972). The vaccines were tested in pairs; each pair on eight guinea-pigs. This was done by injecting two lots on the same guinea-pig in order to make a direct comparison of the reactions and to avoid differences in the response between guinea-pigs (Pierce, Dubos & Schaefer, 1956). Two doses of each lot were used instead of the usual four to reduce to a minimum the influence of injection reactions on each other. The doses were 0.1 ml of the reconstituted undiluted vaccine and 0.1 ml of a 1 : 10 dilution. The three Netherlands lots, P17, P18 and P19, were paired with each other and each of the four Danish lots were paired with lot P17. The latter served as a reference for comparison between the Danish lots and between the Danish and the Netherlands lots. The diameters of the skin-reactions were measured twice a week during six weeks. The maximum responses developed around three weeks (see Fig. 1). The size of the maximum reactions provided better data for analysis of the strength of the vaccines than the time of duration of the reactions (Jensen, 1946), which is difficult to determine. 8
3
7
IO
14
17
21 24
28
31
35
38
42
Time (days)
Fig. 1. The development of the skin reactions on guinea-pigs during 42 days following inoculation of two doses of lot P17 (Netherlands) and of lot K41 (Danish). Each point represents the mean of the diameters of the reactions on eight guinea-pigs.
161
J. L. SIRKS
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AL.
Mouse virulence test
In addition to the Jensen-test, the determination of the virulence of each vaccine was based also on the multiplication of the BCG organisms in the spleens of mice (Pierce, Dubos & Schaefer, 1953, 1956; L&y, Mande, Conge, Fillastre & Orssaud, 1968). Details of our method were published earlier (Sirks, 1973). On three consecutive days the content of one ampoule of each of the seven lots of vaccine was reconstituted and diluted in O.lo/o bovine albumin solution in such a way that the volume of O-2ml to be injected into each mouse contained one hundredth of a human dose. Each preparation was injected intravenously into three 30-35-day-old female mice of the RIV Swiss strain. The test was repeated on three consecutive days one month later. In this way vaccine from six ampoules of each lot was injected into six groups of three mice each. The optimum time to examine the spleens was determined in a series of preliminary experiments with the same lots in which mice were killed after one, three and six weeks. It was then found that the number of the BCG bacteria in the spleens was maximal about three weeks after injection. Therefore, the mice were always killed three weeks after inoculation. Only two of the three mice injected with each lot were killed and the spleens removed. The third mouse in each group served as a replacement in case of an intercurrent death. The number of culturable BCG particles in each spleen was determined as follows: the spleen was ground in a Potter tube in 10 ml of 0.1% bovine albumin solution, then diluted 1 : 10 and 1 : 100; 0.2 ml of each dilution was inoculated in duplicate on Liiwenstein-Jensen medium. Colony counts were made after one month of incubation and the number of culturable particles recovered from each spleen was calculated.
RESULTS Number of culturable particles and stability Table 1 gives the results of the colony counts estimated per human dose of each lot of vaccine after storage at -20 “C and after being held for one month at 37 “C. The Netherlands vaccine had about one-half of the number of culturable particles per human dose as was present in the Danish vaccine. The results of the heat-stability test obtained with each product showed little variations. 1. Number of culturable particles per human dose for three lots of a Netherlands vaccine and four lots of a Danish vaccine after storage at - 20 “C and after one month at 37 “C
TABLE
Culturable particles in thousands Vaccine Netherlands
Lot no.
Storage at -20 “C
After 1 month 37 “c’
Survival (%)
P17
200 336 236
80 184
440 440
200 150 270 160
40 55 43 45 34 63 41
P18 P19
Danish
K32 K41 K46 K48
162
430 390
104
FREEZE-DRIED Guinea-pig
BCG
skin reactivity
VACCINES.
I
test
An example of the reactions of guinea-pigs to the inoculation of two amounts each of P17 and lot K41 is given in Fig. 1. The lesions induced by both vaccines reach their maximum diameter between 17 and 24 days after injection. Similar results were observed with the other lots of vaccine of each product. Table 2 gives the mean diameter of the lot
TABLE
2. Mean diameters of skin reactions three weeks after injection 0.1 ml Pairs of vaccine lots* Pl8 P17 PI8 -i P19 P17 { P19 P17 -C K48 P17 1 K46 P17 -C K41 P17 K32
Undiluted 8.2.f 7.4 8.9 8.4 7.7 8.2 7.5 6.2 7.4 6.7 7.0
5.1 9.6 8.8
of
1 : 10 8.0 6.4 7.7 6.7 5.2 5.9 5.7 4.4 6.5 5.6 5.9 3.6 8.6 7.8
* Each pair was tested on eight guinea-pigs. t Mean diameter (mm) of the eight reactions.
reactions induced by each test dose of each of the paired lots on eight guinea-pigs that were read at three weeks after injection. This time was optimum for evaluation, as shown in Fig. 1. A comparison of the reactivity of each pair of lots tested is shown in Fig. 2. Each point represents the difference between the mean diameter of the reactions induced by one lot on a single guinea-pig and the mean diameter of the reactions induced by the other lot tested on the same guinea-pig. The Netherlands vaccines were more reactogenic than the Danish vaccine. P18 appeared to be the most reactogenic. It induced larger reactions than P17 on seven of eight guinea-pigs and the reactions were larger than or equal to those caused by P19. On the other hand, P17 was more reactogenic than each of the four Danish vaccine-lots. Their comparison with P17 suggests that there was no difference between their reactivity on guinea-pigs. Mouse virulence test
Table 3 gives the calculated number of culturable particles of BCG bacteria per spleen. For each lot of vaccine the geometric mean of individual values appears in the last column. For statistical analysis individual values were transformed to a logarithmic scale. Then the mean value of the logarithm (to base 10) of the number of culturable particles for the two mice in each group was calculated. This provided a figure for each combination of one of the six injection days and each of the seven lots of vaccine. An analysis of variance was applied to these data. The variation between lots of vaccine was split into three parts, viz. the contrast between P and K vaccines, differences between 163
J. L.
SIRKS
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the three lots of P vaccine and differences between the four lots of K vaccine. The results of the analysis are shown in Table 4.
.
5
f 4r 3 . !! 2-• .s I--A 2 Q B 5 -I0g -3--2- . PM-PI7
t.f,r: . as ..
t.
.8 .
. .
? .
0.. Pl6-PI9
Pl7-PI9
P17-K46
P17-K46
PI7-K4l
Pl7-K32
Fig. 2. Comparison of pairs of vaccine lots in groups of eight guinea-pigs. Differences on individual guinea-pigs between mean diamater of skin reactions to two doses of one vaccine lot and mean diameter of reactions to two doses of one other vaccine lot, three weeks after inoculation.
There were rather large differences between individual mice. Nevertheless the figures demonstrate clearly that many more culturable particles are found in the spleen three weeks after injection of one-hundredth of a human dose of the Netherlands (P) vaccine lots than three weeks after injection of the same dose of the Danish (K) vaccine lots. It is also evident that P18 is more virulent for mice than P17 and P19. No systematic differences were detected, neither between P17 and P19 nor among the four lots of K vaccine.
DISCUSSION The foregoing results show that it was possible to prepare a stable and potent freeze-dried vaccine, which meets the W.H.O. Requirements, by cultivation of the strain 1173-P2 in deep culture. Brindle, Griffiths, Holme, Stalker, Burland, Coates & Muggleton (1972) have shown earlier that this is also possible with seed-lot 1331 from Copenhagen and not merely with seed-lot 1077 which is normally used in England for the preparation of vaccine from deep cultures. In this investigation we wanted to compare two types of vaccines and not merely two strains. Therefore we used the vaccine suspensions after reconstruction as recommended for use in man as a starting point for further diluting them to the same extent for testing in animals. This is in contradistinction to the method of several investigators who based their dilution-scheme on equal viable counts or equal dry weights (e.g. Pierce et al., 1956; Benoit & Panisset, 1963 ; Frappier, Portelance, St. Pierre & Panisset, 1971; and others).
The results of our experiments demonstrate that the three vaccine lots prepared by us from the Paris strain all gave larger local vaccination reactions in guinea-pigs and multiplied to a larger extent in spleens of mice than the four vaccine lots prepared in Copenhagen from seed-lot 1331, although the latter had about twice the number of culturable particles per human dose. 164
II.3 32.3 28.2 * 0.24 I.4 0.51
12.5 97.2 29.7 0.27 * 0.46 0.76
20.3 31.3 18.5 * 0.87 0.71 1.6
Group 3 15.9 55.0 13.9 3.0 * 2.6 0.91
Ifs*0 41.6 80.6 9.0 3.5 I.2 2.8
18-l 140.2 107.3 044 4.2 2.0 * the calculated
11.1 91.8 72.9 0.95 12.5 * 0.23
particles in thousands per spleen Test 2 A , c Group 4 Group 5
of culturable
23.6 60.0 5.0 2.3 I.6 0.46 *
Estimated number Test 1 A Group 2
estimate
86.7 39.3 IO.0 6.1 0.23 2-o 4.7
,
16.2 64.6 27.5 1.20 0.96 1.18 0.68
Geometric meant for 12 mice
mice,
if one colony had grown
5.2 72.5 17.9 0.25 9.4 11-l 0.23
Group 6
of BCG in spleens of mice. Estimated number of cuhurable particles in the spleen of individual three weeks after intravenous injection of one-hundredth of a human dose of vaccine
10.2 124.8 36.4 7.0 o-43 I.3 2.3
Group 1
25.7 84.1 39.1 6.6 0.68 I.6 0.71
f
3. Multiplication
* No colonies developed on the media inoculated with the 1 : 100 or 1 : 10 dilutions. t For the counts of spleens from which no growth was obtained, we assigned a value of one-half from the 1 : 10 dilution.
PI7 P18 P19 K32 K41 K46 K48
Vaccine lot
TABLE
J. L. SIRKS
ET AL.
TABLE 4. Analysis of variance, applied to mean values calculated for each of 42 groups of two mice denoted in Table 3 (each mean value regards the logarithm of the estimated number of culturable particles in the spleens of two mice) Source of variation Between days Between vaccine lots -P-lots versus K-lots
Degrees of freedom 5 6 1 2 3
-Between P-lots -Between Residual Total
K-lots 30 41
Sum of squares
Mean square
1.161 24.356 23.006 l-114 0.236 4.244 29.760
0.232
1.64
N.S.
23.006 0.557 0.079 0.141
162.6 3.94 0.56
P
F-ratio
Significancy
These findings confirm the experience of other investigators that the viable count as such is not a sufficient criterion for the comparison of the quality of BCG vaccines of different origins (Levy et al., 1968; Frappier et al., 1971). The viable count, however, has value as a check on the degree of consistency in the production of consecutive lots of a vaccine prepared from the same strain in the same way and as a check on the stability of the vaccine during freeze-drying and during storage at different temperatures (W.H.O. Expert Committee on Biological Standardization, 1966). For the comparison of vaccines, prepared in different ways from different substrains, animal experiments are necessary for potency and safety evaluation. These tests are also of value in the control of routine production of successive lots of the same product. This was demonstrated by the fact that a slight but distinct difference in potency between the Netherlands lot P18 and the two other lots P17 and P19 could be found in this way. The Jensen-test is recommended in the W.H.O. Requirements for dried BCG vaccine (1966) under ‘Safety-tests’. It may, however, serve simultaneously as a potency test. If a vaccine induces larger reactions in guinea-pigs than a reference preparation one might expect that it would be able also to elicit a larger and longer lasting Mantoux-reactivity and possibly give better protection in man (Levy et al., 1968; Jespersen, 1971; Guld, 1971). On the other hand, if a vaccine induces significantly larger reactions than a reference preparation with an acceptable reactogenicity for man it might cause undesirable vaccination reactions. The test on the multiplication of the BCG bacteria in the spleen of mice is mainly a potency test. Dubos & Pierce (1956) concluded earlier that ‘there is no doubt that immunity is an expression of the ability of the BCG organisms to multiply in the body of the vaccinated individuals’. In general differences between two vaccines with respect to their rate of multiplication in low doses.
in the spleen of mice are observed only if they are injected
The best time for the determination of the number of culturable particles in the spleens is when multiplication is maximal. We found that for most vaccines this time was three weeks after intravenous injection. In this way we found for the P and K vaccines a good correlation between the results of the mouse potency test and the Jensen-test. The greater virulence of the P vaccines in these experiments may be due chiefly to the
substrain from which it has been prepared. Ladefoged, Bunch-Christensen (1970) found that a liquid
& Guld
vaccine made from seed-lot 1173-P2 gave better protection
in
low doses in bank voles against virulent tubercle bacilli tban a liquid vaccine which was 166
FREEZE-DRIED
BCG VACCINES.
I
prepared from the Danish seed-lot 1331 in exactly the same way. It cannot be excluded, however, that besides the properties of the strain the method of preparation has some influence on the potency of the vaccine. Homogenization of the bacterial mass by grinding in a ball mill is avoided by application of the deep-culture technique. This may result in a reduced number of killed or damaged mycobacteria. The two types of vaccines we studied did not only differ in the substrain used, the method of cultivation and the method of harvesting and resuspending the bacteria but also in the type of fluid carrier for freezedrying. Any one of these factors alone or combined may have contributed to differences between the Netherlands and the Danish vaccines. None of these factors, however, can account for the fact that one of the three P vaccines was somewhat more virulent than the others. The results of the field trial, to be published in the second part of this communication (Sekhuis et al., 1974) show that the two types of vaccine induced in man post-vaccinal Mantoux- and vaccination reactions which were in agreement with their reactogenicity in guinea-pigs and their multiplication rate in mice as described in this paper. Acknowledgements We wish to thank Dr K. Bunch-Christensen, Statens Seruminstitut, for supplying the Danish vaccines; Dr F. M. L&y, International Children’s Centre Paris, for the testing of seed-lot 1173-P3 in mice; Dr Margaret Pittman, Washington D.C., U.S.A., for helpful comments and criticism of the manuscript; Mr R. W. Mendels, Mr P. van Putten and Mr P. Vonk for their technical assistance.
REFERENCES Benoit, J. L. & Panisset, Mi (1963). Survie et multiplication du BCG et du bacille tuberculeux chez la souris. IV. Acta Tuberculosea et Pneumologica Scandinavica 43, 125-136. Brindle, T. W., Griffiths, M. I., Holme, T., Stalker, R., Burland, W. L., Coates, G. A. & Muggleton, P. W. (1972). A trial to compare reactions and responses to BCG vaccine prepared from Copenhagen 1331 and Glaxo 1077 strains. Tubercle, London 53, 100-105. Dubos, R. J. & Pierce, C. H. (1956). Differential characteristics in vitro and in vivo of several substrains of BCG. IV. Immunizing effectiveness. American Review of Tuberculosis and Pulmonary Diseases 74, 699-717. Frappier, A., Portelance, V., St. Pierre, J. & Panisset, M. (1971). BCG-strains: characteristics and relative efficacy. In: Status of Immunization in Tuberculosis in 1971. Report of a conference on progress to date, future trends and research needs. DHEW Publication no. (NIH) 72-68, pp. 157-178. Guld, J. (1971). BCG as an immunizing agent. In: Status of Immunization in tuberculosis in 1971. Report of a conference on progress to date, future trends and research needs. DHEW Publication no. (NIH) 72-68, pp. 149-156. Jensen, K. A. (1946). Practice of the Calmette vaccination. Acta Tuberculosea Scandinavica 20, l-45. Jespersen, A. (1971). The potency of BCG-vaccines determined on animals. Thesis, Copenhagen, Chapter 5, pp. 82-83. Ladefoged, A., Bunch-Christensen, K. & Guld, J. (1970). The protective effect in bank voles of some strains of BCG. Bulletin of the World Health Organization 43, 71-90. L&y, F. M., Mande, R., Conge, G., Fillastre, C. & Orssaud, E. (1968). Perspectives for BCG standardization. Advances in Tuberculosis Research Vol. 16, Chapter IV-3, pp. 124-128. Cane& G., Birkhluser, H., & Block, H., eds. New York: Karger, Basel. Pierce, C. H., Dubos, R. J. & Schaefer, W. B. (1953). Multiplication and survival of tubercle bacilli in the organs of mice. Journal of Experimental Medicine 97, 189-206. 167
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Pierce, C. H., Dubos, R. J. & Schaefer, W. B. (1956). Differential characteristics in vitro and in wivo of several substrains of BCG. III. Multiplication and survival in viuo. American Review of Tuberculosis and Pulmonary Diseases 74, 683-698. Schmidt-Thorn& J., Mayer, A. & Schone, H. H. (1962). Zur Chemie eines neuen Plasmaexpanders. Arzneimittelforschung 12, 378-380. Schwick, G. & Freund, U. (1962). Immunologische Untersuchungen mit Haemaccel. Deutsche Medizinische Wochenschrift 87, 737-741. Sekhuis, V. M., Bleiker, M. A., Sirks, J. L. & Cohen, H. (1974). Comparison of two types of freeze-dried BCG vaccine products. II. Results of vaccination of men. Journal of Biological Standardization 2, 169-181. Sirks, J. L. (1973). Controle van BCG-vaccin door bepaling van de mate van vermenigvuldiging in muizen. Verslagen en Mededelingen betreffende de Volksgezondheid 26, 190-193. Sirks, J. L. & Cohen, H. (1972). Controle van BCG-vaccin volgens Jensen door intracutane injecties bij cavia’s. Verslagen en Mededelingen betreffende de Volksgezondhe-id 24, 206212. Sirks, J. L., Cohen, H. & Smith, L. (1971). Production of freeze-dried BCG-vaccine. Symposia Series in Immunobiological Standardization 17, 163-168. Ungar, J., Muggleton, P. W., DudIey, J. A. R. & Griffiths, M. I. (1962). Preparation and properties of a freeze-dried BCG-vaccine of increased stability. British MedicalJournal II, 1086-1089. W.H.O. Expert Committee on Biological Standardization (1966). Requirements for dried BCG-vaccine. W.H.O. Technical Report Series no. 329, Annex 1, pp. 25-48.
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