- Email: [email protected]
Evaluation of the stability of dried BCG vaccine
92
Tuhtwlr.
EVALUATION
OF THE STABILITY
(1972). 53, 92
OF DRIED BCG VACCINE
By D. WRIGHTand P. W. MUGGLETON Jiiomthe GIaxo Laboratories Ltd., Greenford, Middlexx and MARGARETI. GRIFF~THS from the Institute
of Child Health,
University
of Birmingham
SUMMARY Dried BCG vaccine, prepared by the method of Ungar and others (1962) (deep culture growth in glycerol-free liquid medium, freeze dried in dextran-glucose solution) has been tested for stability on storage at refrigerator temperature (0-5”C), room temperature (1822°C) and elevated temperature (37°C). Vaccine incubated at 37°C for 56 days retained sufficient viabiiity (about 4 per centj to give, when tested in schooi chiidren, vaccination lesions and tuberculin sensitivities which were indistinguishable from those given by refrigerator-stored, control vaccine. It was concluded that heat stability, as judged by these clinical and laboratory findings, is sufficient for convenient routine use of the vaccine in tropical countries.
Du vaccin BCG set, prepare par la mtthode de Ungar et ~011. (1962) (cultures en profondeur dans un milieu liquide ne contenant pas de glycerol, lyophilist dans une solution de dextrane glucose) a Ctt ttudie quant a sa stabilite lors de sa conservation a la temperature du refrigerateur (O-5’ C), a la temperature ambiante (18-22” C) et a une temperature tlevee (37” C). Du vaccin laisse a 37” C pendant 56 jours avait conserve une viabilite suffisante (a peu pres 40 %) pour donner, au tours dune vaccination a des Ccoliers, des lesions de vaccination et des reactions de sensibilite a la tuberculine, qui n’ttaient pas differentes de celles don&es par un vaccin temoin garde au refrigerateur. 11 s’ensuit que la stabilite a la chaleur, comme le montrent ces etudes cllniques et de laboratoires, est suffisante pour que ce vaccin soit utilise en pratique de routine dans les pays tropicaux.
RESUMEN Vacuna BCG seca, preparada por el metodo de Ungar y otros (1962) (crecimiento en profundidad en medio liquid0 sin glicerina, secado por congelation dextran-glucosa) fuC probada en cuanto a estabilidad almacenandola a temperatura de heladera (O-5’%), a temperatura ambiente (1822°C) y a temperatura elevada (37°C). La vacuna incubada a 37°C durante 56 dias conservo suficiente vitalidad (alrededor de1 40%) para producir en nifios escolares reacciones vaccinales y sensibilizacion a la tuberculina andlogas a las producidas por una vacuna control, almacenada en heladera. Se concluye que el calor estable, de acuerdo con estos hallazgos, es suficiente para el uso rutinario de las vacunas en regiones tropicales.
STABILITY
OF BCG
93
ZUSAMMENFASSUNG
Getrocknete BCG-Vakzine, die nach der Methode von Ungar und Mitarbeitern (1962) hergestellt wurde (tiefes Kulturwachstum in glyzerinfreiem fltissigem Niihrboden, gefriergetrocknet in einer Dextran-Glukose-Losung) wurde auf Haltbarkeit bei Lagerung unter Kiihlschranktemperatur (0-5C), Zimmertemperatur (18-22°C) und bei erhohter Temperatur (37°C) getestet. Vakzine, die 56 Tage lang einer Temperatur von 37°C ausgesetzt war, enthielt danach immer noch eine ausreichende Anzahl (etwa 40 Prozent) lebender Keime. Mit einem solchen Impfstoff entwickelten sich Impflasionen und Tuberkulinempfindlichkeit in einer Weise, die nicht von denen zu unterscheiden waren, die entstanden, wenn eine im Ktihlschrank aufbewahrte Kontrollvakzine verwendet wurde. Die Hitzestabilitat, die auf Grund dieser Untersuchungen zu unterstellen ist, erscheint groB genug, urn die routinemafiige Verwendung dieser Vakzine in tropischen Landern zu rechtfertigen. Introduction
Dried BCG vaccine, much of which is used in tropical countries, should be made by a process which gives a good degree of stability. When sufficiently heat stable, the vaccine can be transported more easily without the need for refrigerated conditions; moreover, the consequences of accidental exposure to higher ambient temperatures, with resultant diminution of immunising potency, are minimised. A heat stable vaccine was described by Ungar and others (1962) in which the BCG organisms were freeze-dried in a drying medium consisting of dextran/glucose solution after growing them in a medium which did not contain glycerol. Considerable experience has since been gained concerning the heat stabiiity of this vaccine and this paper gives the resuits of keeping tests, inciuding tests of vaccine stored at higher temperatures. These tests are based not only on laboratory viability counts but also on tests of tuberculin conversion in children. Methods Vaccines used
Full scale production batches of dried BCG vaccine were made by the method of Ungar and others (1962). This essentially consists of growing the organisms _mediu_m without ~-v---------- in a iioluid glycerol but containing 0.025 per cent of Triton W.R. 1339. In the presence of this wetting agent, the organisms produce a diffuse growth in the depth of the medium. They are harvested by centrifuging and freeze-dried in a medium consisting of a solution of dextran (8 per cent) and glucose (7.5 per cent) in distilled water. The vaccine is prepared in neutral glass ampoules which are sealed in vacua. Viability counts
Viability counts were done by a technique similar to that of Miles, Misra and Irwin (1938). Serial ten-fold dilutions of the reconstituted vaccine were plated on to the surface of oleic acid albumin medium containing 5 per cent human blood. Up to 10 drops were plated on to each petri dish using a 50 drops per ml platinum-tipped pipette. The petri dishes were sealed in polythene bags to prevent desiccation during 14 days incubation at 37°C. The colonies of BCG were then enumerated under a stereo-binocular plate microscope using tangential illumination of the colonies. Viability counts were expressed as the number of culturable particles per ml of reconstituted vaccine. Results
Tests were carried out on a number of vaccine batches, selected at random, in order to check that the viability of each vaccine batch was satisfactorily maintained during storage at 0-5°C. The tests were continued for periods in excess of the accepted total ‘life’of the vaccine of 2 years. The.
94
\VRIGHT
AND
OTHERS
results are shown in Figure 1. They indicate that the number of viable BCG organisms in the dried vaccine decreases by approximately 10 per cent per annum during storage at 0-5°C. A test of stability to an elevated temperature was also done on the batches of vaccine. Viability counts were made on samples which had been stored at 37°C for I month and these were compared with control samples stored at 0-5OC during the same period. The results on 100 batches are shown as histograms in Figure 2. They indicate that the vaccine was relatively stable at the elevated temperature, some 40-45 per cent of the organisms remaining viable after exposure to 37°C for 1 month. A further test for heat stability, similar to that suggested in the World Health Organisation T.B. Tech. Guide/67.6, page 17, was also done. Samples of the batches of vaccine were transferred from the refrigerator to storage at two elevated temperatures, 18°C and 37°C at intervals over a period % ^.._. .. .. . MJKVIVAL.
-
4
B
I2
STORAGE
16
20
24
28
32
Y=98~5coe2x
36
TIME MONTHS FIG. 1
Stability of dried BCG vaccine O”-5°C.
of 8 weeks, such that at the end of the period samples had been stored for 2, 4, 6 and 8 weeks. Viable counts were then done on all samples in one session. The average results of this test on three batches of vaccine are shown in Figure 3. The viability of the dried vaccine was reduced by approximately 10 per cent per month by exposure to average U.K. room temperatures, i.e. approximately 18°C and by approximately one-third per month by exposure to 37°C. It thus seemed that after exposure for 1 month to a temperature of 37°C sufficient viable organisms would probably remain to achieve a satisfactory vaccination of children. To test this point, two clinical trials were organised. Clinical Trials
The first was carried out in 1966-67 with the object of establishing whether freeze dried BCG vaccine (intradermal type) remained fully immunising after exposure to 37°C for 1 month.
No.
EK”ES 2
I
16
14
i
% SURVIVAL Heat stability
37k
28DAYS
FIG. 2 of dried BCG vaccine.
100 Batches
%
SURVIVAL
STORAGE Stability
TIME
WEEKS
FIG. 3 of dried BCG vaccine.
0.f
1
1.P
82
9s
9.L
0.8
9.9
6.5
3,Lf 3”ZZ 3.>s-o
nrngmdurar
YULLI CUOLU
.IO 9 JO
J&LlOlS
~a~au~e!p as~a~sue~~ B se pauyapSTuo!ysa~ ,anqsod,
v*
.alnp.xadrual pale~ala aq$ 01 aJnsodxa Lq papa@? 6pu1~~~~u~!sSCMuo!sal uoyx1!331?~ aq) JO az!s ayl JOU uog%a.t xnolue~ aq130 az!s ayl .rayl!a~ ,*uo!s.~a~uo~ UgnXIaqn) lua:, lad 001 a&3 ‘y~uow 1 ~03 3,LE 01 amsodxa .ral3~ uaAa ‘aupDeh 30 ~$01qloq JEql a~wpu~ LayJ y7 a&i!~ u! sma.15?o~s!yse u~oys ain suoy3ea.I pi?301aql30 azrs ayl pw ihgsal ugnD.raqnl ayl30 slInsal ayl aq 0) palap!suoD SI?Mpa3npold UoyUIpu! 30 .Ia]aun?!p ueaw aq$ put? ‘Jua3lad ()()I SEMuo!s.IaAuo3 .pa.nw2ar_uset uo!saI uogeup~w ayl 30 az!s ayl ‘auop svM Isal ugn3laqn~ ayl se auy auws ayl IV *atyeA aqj %1!12!8layt? syaaM 6 auop SBM(aBp!.tqlCaMadd 30 nl 01) Isal xnoy_wM dn -MOIIOJv ‘p!ollap ayl30 uoyasu! ayl meUIJ~ ayl uo alpaau pue a%r!.& Lq LI[aur~apw~u! ua@ %u!aq au!Xt?h 30 1u11.o ‘ualpIy3 1ooy3s ()9-& 30 sdno.tS 30 uopup3e~ aq$ u! pasn aJaM ~$01au!Xen ayJ_ ./C~aayadsa~ lua3 lad ~9 PUB JUW lad zp Lq ywour I .103~,LE 01 alnsodxa Icq pmnpal actaMZLL pue 0LL sayDl,,aqwyy alwpu! ‘I a1qvl u! uMoys ‘swno:, L)!yqyh ayl .auop alah PUBIsal yea 30 saInodtuv syuno3 /(yyqt?!a ‘auq atuBs aqy JB ‘pue palsal Ic~p+uy aJaM 101IO.IJUO~ /(luaML ‘a,~-0 $r! paloval aJaM saldtues lsal ‘uogeqnou! layv *(IO.IJUOD) ~~g-0 )e pauyal Zkyaq 701Jaylo aq2 ‘@al) sdep 82 103 sOLE v paloIs SBMqwq yDt?a30 101au0 *(zLLput! OLLpaiaqwnu) saycwq aupX?A f)sg pXu.tapt?Jlu~z 30 yX?a u10.13paldtuw alaM saInoduw 0s 30 ~$01 OML
6.5
~
1.P
P,f
I
s.1
I
(sapodura
01 jo
sdeP 82 1OJ3,:Lf /
WJ1~0~)
30 s-o
a.07 spsa;y)
w~dlll
96
STABILITY Mantow
OF
97
BCG
tests (10 TU PPD)
Local reactlOllS
/
average reaction diametet=20~5mm
!.
r
average reaction diameter=21
average lesson diameter=8
Batch No 770E (after 28 days at 37°C) 1
9mm
Batch No 772 -IstoredatO SOCiv
5
15
average reaction dlameter=19
3mm
I
dverage lesmn dbameter=8
9mm
7mm
D i
Batch No 772E (after 28 days at 37°C) r
average reactlo” diameter=19
mm’s
1
lmm
mm’5
FIG. 4 Mantoux reactions and vaccination lesions in school children 9 weeks after vaccination with heated (37°C for 28 days) BCG vaccine compared to refrigerator-stored control vaccine.
The second clinical trial was begun in August 1967. One standard batch of intradermal BCG vaccine (Batch B833) was chosen and 30 ampoule lots were held at 0-5°C 22°C and 37°C for (a) 28 days, and (b) 56 days. All lots were then restored at O-5%. Ten ampoules of each lot were tested for viability, and 20 ampoules of each lot were used for a clinical test. The viability results are shown in Table II. Exposure for 28 days to 22°C and 37°C resulted in viable count reductions of 13 per cent and 46 per cent respectively and exposure for 56 days to 22°C and 37°C resulted in viable count reductions of 26 per cent and 63 per cent. The vaccine samples were tested clinically as before in groups of 46-70 children. Mantoux tests (10 TU of PPD Weybridge) were done at 14 weeks. The results are shown in Table III. Storage of the vaccine for 28 and 56 days at 22°C and 37°C did not significantly affect the
98
WRIGHT
TABLE III.-SECOND
AND
CLINICAL TEST OF HEAT STABILITY OF
OTHERS
BCG
DRIED VACCINE BATCH B833: TUHEKNLIN
TESI-s
AT 14 WEEKS
Vaccine stored: Period
u-5 C‘
37’C‘
28 days
64
66
hl
56 days
69
70
46
28 days
I00
Number of subjects -___
100
:
100
Per cent positive* to 10 TU of PPD 56 days ___.___
98.6
100 ___._~__
~
-- --,
28 days
1
18.3
56 days
~
18.7
___.~
100
19.5
20.0
19.1
18.7
~~
Mean diameter of induration in mm
*Transverse diameter of 6 mm or more.
mean diameters of induration obtained to 10 TU of PPD in the Mantoux tests. The mean diameters were all very similar to those resulting from the control lot of vaccine which had been stored at o-5°C. Following this finding, and to establish whether the tuberculin sensitivity was sustained, the school children who had received the vaccine lots which had been stored for 56 days at 0-5°C 22°C and 37°C were tuberculin tested again 14 months after vaccination. The results are summarised in Table IV. The mean diameters of induration were of the same order at 14 months as at 14 weeks post-vaccination. Conversion was 100 per cent from the vaccines which had been stored at 22°C and at 37°C and 96.7 per cent from the vaccine which had been stored at 0-5°C. Discussion Heat stable BCG vaccine is of obvious advantage for successful vaccination in tropical countries. Surface transport without refrigeration is possible and, more important, accidental or unrealised exposure to ambient temperatures for short periods does not destroy the potency of such vaccines. Many years of experience in the manufacture and distribution of vaccine to overseas countries TABLE IV.-SECOND
CLINICAL TEST OF HEAT STABILITY OF BCG DRIED VACCINE BATCH
14
B833 : TUBERCULIN
TESTS AT
MONTHS
--___-~~...~~~~~.._
_
_~____
I
Vaccine stored 56 days at :
o-5°C
/
22°C
37°C
57
39
-__
Number of cases --_ Per cent positive* to 10 TU of PPD at 14 months post-vaccination
61 96.7
Mean diameter of induration in mm *Transverse diameter of 6 mm or more.
100
100
STABILITY
OF BCG
99
have shown that such episodes as storage in unrefrigerated sheds awaiting customs clearance or delayed surface or air transport cannot be entirely avoided. In this paper we have shown that BCG vaccine, made by the process of Ungar and others (1962), will withstand storage at refrigerator temperatures for at least 2 years, temperate room temperature for at least 8 weeks and tropical room temperature (37°C) for 4-8 weeks without an excessive fall in viability. After storage at 37°C for periods up to 56 days, tests on school children showed that the remaining viable organisms were sufficient to achieve a full degree of tuberculin allergy to an intensity similar to that produced by unheated vaccine. Moreover, when the tuberculin tests were done 14 months later, the intensity of the tuberculin allergy was undiminished. Previous experience suggests that this is a good indication of a successful vaccination since, if the effect of the vaccination had been transitory, allergy would start to wane by this time. It therefore seems that vaccine which has been accidentally exposed to elevated temperatures for a few weeks, even in tropical countries, can still be used satisfactorily for vaccination. It is clear, however, that no vaccine will withstand abuse of its recommended storage conditions repeatedly; refrigeration should be used as a routine measure. This is not always realised and it is suggested that caution should be exercised in labelling BCG vaccine ‘heat stable’ in case this leads to carelessness in storing the vaccine. Despite ‘heat stability’ its effective life at elevated temperatures is reduced to weeks rather than years if the recommended refrigerated storage is not adopted. Thanks are due to Mrs A. Stanbrook and Mr J. A. R. Dudley who assisted with the laboratory work, and to Dr J. F. Galloway and Dr G. Ramage for their co-operation in permitting the additional follow-up examinations involved in the clinical evaluation. REFERENCES MILES,A. A., MICRA,S. S. & IRWIN, J. 0. (1938). The estimation
of the bactericidal power of the blood. Journal of
Hvaiene, Cambridpe, 38, 732.
UNGA~~J., MUGGLETO~P. W., DUDLEY,J. A. R. & GRIFFITHS, M. I. (1962). Preparation
dried B.C.G. vaccine of increased stability. British Medical Journal, 2, 1086.
and properties of a freeze-
Tuhtwlr.
EVALUATION
OF THE STABILITY
(1972). 53, 92
OF DRIED BCG VACCINE
By D. WRIGHTand P. W. MUGGLETON Jiiomthe GIaxo Laboratories Ltd., Greenford, Middlexx and MARGARETI. GRIFF~THS from the Institute
of Child Health,
University
of Birmingham
SUMMARY Dried BCG vaccine, prepared by the method of Ungar and others (1962) (deep culture growth in glycerol-free liquid medium, freeze dried in dextran-glucose solution) has been tested for stability on storage at refrigerator temperature (0-5”C), room temperature (1822°C) and elevated temperature (37°C). Vaccine incubated at 37°C for 56 days retained sufficient viabiiity (about 4 per centj to give, when tested in schooi chiidren, vaccination lesions and tuberculin sensitivities which were indistinguishable from those given by refrigerator-stored, control vaccine. It was concluded that heat stability, as judged by these clinical and laboratory findings, is sufficient for convenient routine use of the vaccine in tropical countries.
Du vaccin BCG set, prepare par la mtthode de Ungar et ~011. (1962) (cultures en profondeur dans un milieu liquide ne contenant pas de glycerol, lyophilist dans une solution de dextrane glucose) a Ctt ttudie quant a sa stabilite lors de sa conservation a la temperature du refrigerateur (O-5’ C), a la temperature ambiante (18-22” C) et a une temperature tlevee (37” C). Du vaccin laisse a 37” C pendant 56 jours avait conserve une viabilite suffisante (a peu pres 40 %) pour donner, au tours dune vaccination a des Ccoliers, des lesions de vaccination et des reactions de sensibilite a la tuberculine, qui n’ttaient pas differentes de celles don&es par un vaccin temoin garde au refrigerateur. 11 s’ensuit que la stabilite a la chaleur, comme le montrent ces etudes cllniques et de laboratoires, est suffisante pour que ce vaccin soit utilise en pratique de routine dans les pays tropicaux.
RESUMEN Vacuna BCG seca, preparada por el metodo de Ungar y otros (1962) (crecimiento en profundidad en medio liquid0 sin glicerina, secado por congelation dextran-glucosa) fuC probada en cuanto a estabilidad almacenandola a temperatura de heladera (O-5’%), a temperatura ambiente (1822°C) y a temperatura elevada (37°C). La vacuna incubada a 37°C durante 56 dias conservo suficiente vitalidad (alrededor de1 40%) para producir en nifios escolares reacciones vaccinales y sensibilizacion a la tuberculina andlogas a las producidas por una vacuna control, almacenada en heladera. Se concluye que el calor estable, de acuerdo con estos hallazgos, es suficiente para el uso rutinario de las vacunas en regiones tropicales.
STABILITY
OF BCG
93
ZUSAMMENFASSUNG
Getrocknete BCG-Vakzine, die nach der Methode von Ungar und Mitarbeitern (1962) hergestellt wurde (tiefes Kulturwachstum in glyzerinfreiem fltissigem Niihrboden, gefriergetrocknet in einer Dextran-Glukose-Losung) wurde auf Haltbarkeit bei Lagerung unter Kiihlschranktemperatur (0-5C), Zimmertemperatur (18-22°C) und bei erhohter Temperatur (37°C) getestet. Vakzine, die 56 Tage lang einer Temperatur von 37°C ausgesetzt war, enthielt danach immer noch eine ausreichende Anzahl (etwa 40 Prozent) lebender Keime. Mit einem solchen Impfstoff entwickelten sich Impflasionen und Tuberkulinempfindlichkeit in einer Weise, die nicht von denen zu unterscheiden waren, die entstanden, wenn eine im Ktihlschrank aufbewahrte Kontrollvakzine verwendet wurde. Die Hitzestabilitat, die auf Grund dieser Untersuchungen zu unterstellen ist, erscheint groB genug, urn die routinemafiige Verwendung dieser Vakzine in tropischen Landern zu rechtfertigen. Introduction
Dried BCG vaccine, much of which is used in tropical countries, should be made by a process which gives a good degree of stability. When sufficiently heat stable, the vaccine can be transported more easily without the need for refrigerated conditions; moreover, the consequences of accidental exposure to higher ambient temperatures, with resultant diminution of immunising potency, are minimised. A heat stable vaccine was described by Ungar and others (1962) in which the BCG organisms were freeze-dried in a drying medium consisting of dextran/glucose solution after growing them in a medium which did not contain glycerol. Considerable experience has since been gained concerning the heat stabiiity of this vaccine and this paper gives the resuits of keeping tests, inciuding tests of vaccine stored at higher temperatures. These tests are based not only on laboratory viability counts but also on tests of tuberculin conversion in children. Methods Vaccines used
Full scale production batches of dried BCG vaccine were made by the method of Ungar and others (1962). This essentially consists of growing the organisms _mediu_m without ~-v---------- in a iioluid glycerol but containing 0.025 per cent of Triton W.R. 1339. In the presence of this wetting agent, the organisms produce a diffuse growth in the depth of the medium. They are harvested by centrifuging and freeze-dried in a medium consisting of a solution of dextran (8 per cent) and glucose (7.5 per cent) in distilled water. The vaccine is prepared in neutral glass ampoules which are sealed in vacua. Viability counts
Viability counts were done by a technique similar to that of Miles, Misra and Irwin (1938). Serial ten-fold dilutions of the reconstituted vaccine were plated on to the surface of oleic acid albumin medium containing 5 per cent human blood. Up to 10 drops were plated on to each petri dish using a 50 drops per ml platinum-tipped pipette. The petri dishes were sealed in polythene bags to prevent desiccation during 14 days incubation at 37°C. The colonies of BCG were then enumerated under a stereo-binocular plate microscope using tangential illumination of the colonies. Viability counts were expressed as the number of culturable particles per ml of reconstituted vaccine. Results
Tests were carried out on a number of vaccine batches, selected at random, in order to check that the viability of each vaccine batch was satisfactorily maintained during storage at 0-5°C. The tests were continued for periods in excess of the accepted total ‘life’of the vaccine of 2 years. The.
94
\VRIGHT
AND
OTHERS
results are shown in Figure 1. They indicate that the number of viable BCG organisms in the dried vaccine decreases by approximately 10 per cent per annum during storage at 0-5°C. A test of stability to an elevated temperature was also done on the batches of vaccine. Viability counts were made on samples which had been stored at 37°C for I month and these were compared with control samples stored at 0-5OC during the same period. The results on 100 batches are shown as histograms in Figure 2. They indicate that the vaccine was relatively stable at the elevated temperature, some 40-45 per cent of the organisms remaining viable after exposure to 37°C for 1 month. A further test for heat stability, similar to that suggested in the World Health Organisation T.B. Tech. Guide/67.6, page 17, was also done. Samples of the batches of vaccine were transferred from the refrigerator to storage at two elevated temperatures, 18°C and 37°C at intervals over a period % ^.._. .. .. . MJKVIVAL.
-
4
B
I2
STORAGE
16
20
24
28
32
Y=98~5coe2x
36
TIME MONTHS FIG. 1
Stability of dried BCG vaccine O”-5°C.
of 8 weeks, such that at the end of the period samples had been stored for 2, 4, 6 and 8 weeks. Viable counts were then done on all samples in one session. The average results of this test on three batches of vaccine are shown in Figure 3. The viability of the dried vaccine was reduced by approximately 10 per cent per month by exposure to average U.K. room temperatures, i.e. approximately 18°C and by approximately one-third per month by exposure to 37°C. It thus seemed that after exposure for 1 month to a temperature of 37°C sufficient viable organisms would probably remain to achieve a satisfactory vaccination of children. To test this point, two clinical trials were organised. Clinical Trials
The first was carried out in 1966-67 with the object of establishing whether freeze dried BCG vaccine (intradermal type) remained fully immunising after exposure to 37°C for 1 month.
No.
EK”ES 2
I
16
14
i
% SURVIVAL Heat stability
37k
28DAYS
FIG. 2 of dried BCG vaccine.
100 Batches
%
SURVIVAL
STORAGE Stability
TIME
WEEKS
FIG. 3 of dried BCG vaccine.
0.f
1
1.P
82
9s
9.L
0.8
9.9
6.5
3,Lf 3”ZZ 3.>s-o
nrngmdurar
YULLI CUOLU
.IO 9 JO
J&LlOlS
~a~au~e!p as~a~sue~~ B se pauyapSTuo!ysa~ ,anqsod,
v*
.alnp.xadrual pale~ala aq$ 01 aJnsodxa Lq papa@? 6pu1~~~~u~!sSCMuo!sal uoyx1!331?~ aq) JO az!s ayl JOU uog%a.t xnolue~ aq130 az!s ayl .rayl!a~ ,*uo!s.~a~uo~ UgnXIaqn) lua:, lad 001 a&3 ‘y~uow 1 ~03 3,LE 01 amsodxa .ral3~ uaAa ‘aupDeh 30 ~$01qloq JEql a~wpu~ LayJ y7 a&i!~ u! sma.15?o~s!yse u~oys ain suoy3ea.I pi?301aql30 azrs ayl pw ihgsal ugnD.raqnl ayl30 slInsal ayl aq 0) palap!suoD SI?Mpa3npold UoyUIpu! 30 .Ia]aun?!p ueaw aq$ put? ‘Jua3lad ()()I SEMuo!s.IaAuo3 .pa.nw2ar_uset uo!saI uogeup~w ayl 30 az!s ayl ‘auop svM Isal ugn3laqn~ ayl se auy auws ayl IV *atyeA aqj %1!12!8layt? syaaM 6 auop SBM(aBp!.tqlCaMadd 30 nl 01) Isal xnoy_wM dn -MOIIOJv ‘p!ollap ayl30 uoyasu! ayl meUIJ~ ayl uo alpaau pue a%r!.& Lq LI[aur~apw~u! ua@ %u!aq au!Xt?h 30 1u11.o ‘ualpIy3 1ooy3s ()9-& 30 sdno.tS 30 uopup3e~ aq$ u! pasn aJaM ~$01au!Xen ayJ_ ./C~aayadsa~ lua3 lad ~9 PUB JUW lad zp Lq ywour I .103~,LE 01 alnsodxa Icq pmnpal actaMZLL pue 0LL sayDl,,aqwyy alwpu! ‘I a1qvl u! uMoys ‘swno:, L)!yqyh ayl .auop alah PUBIsal yea 30 saInodtuv syuno3 /(yyqt?!a ‘auq atuBs aqy JB ‘pue palsal Ic~p+uy aJaM 101IO.IJUO~ /(luaML ‘a,~-0 $r! paloval aJaM saldtues lsal ‘uogeqnou! layv *(IO.IJUOD) ~~g-0 )e pauyal Zkyaq 701Jaylo aq2 ‘@al) sdep 82 103 sOLE v paloIs SBMqwq yDt?a30 101au0 *(zLLput! OLLpaiaqwnu) saycwq aupX?A f)sg pXu.tapt?Jlu~z 30 yX?a u10.13paldtuw alaM saInoduw 0s 30 ~$01 OML
6.5
~
1.P
P,f
I
s.1
I
(sapodura
01 jo
sdeP 82 1OJ3,:Lf /
WJ1~0~)
30 s-o
a.07 spsa;y)
w~dlll
96
STABILITY Mantow
OF
97
BCG
tests (10 TU PPD)
Local reactlOllS
/
average reaction diametet=20~5mm
!.
r
average reaction diameter=21
average lesson diameter=8
Batch No 770E (after 28 days at 37°C) 1
9mm
Batch No 772 -IstoredatO SOCiv
5
15
average reaction dlameter=19
3mm
I
dverage lesmn dbameter=8
9mm
7mm
D i
Batch No 772E (after 28 days at 37°C) r
average reactlo” diameter=19
mm’s
1
lmm
mm’5
FIG. 4 Mantoux reactions and vaccination lesions in school children 9 weeks after vaccination with heated (37°C for 28 days) BCG vaccine compared to refrigerator-stored control vaccine.
The second clinical trial was begun in August 1967. One standard batch of intradermal BCG vaccine (Batch B833) was chosen and 30 ampoule lots were held at 0-5°C 22°C and 37°C for (a) 28 days, and (b) 56 days. All lots were then restored at O-5%. Ten ampoules of each lot were tested for viability, and 20 ampoules of each lot were used for a clinical test. The viability results are shown in Table II. Exposure for 28 days to 22°C and 37°C resulted in viable count reductions of 13 per cent and 46 per cent respectively and exposure for 56 days to 22°C and 37°C resulted in viable count reductions of 26 per cent and 63 per cent. The vaccine samples were tested clinically as before in groups of 46-70 children. Mantoux tests (10 TU of PPD Weybridge) were done at 14 weeks. The results are shown in Table III. Storage of the vaccine for 28 and 56 days at 22°C and 37°C did not significantly affect the
98
WRIGHT
TABLE III.-SECOND
AND
CLINICAL TEST OF HEAT STABILITY OF
OTHERS
BCG
DRIED VACCINE BATCH B833: TUHEKNLIN
TESI-s
AT 14 WEEKS
Vaccine stored: Period
u-5 C‘
37’C‘
28 days
64
66
hl
56 days
69
70
46
28 days
I00
Number of subjects -___
100
:
100
Per cent positive* to 10 TU of PPD 56 days ___.___
98.6
100 ___._~__
~
-- --,
28 days
1
18.3
56 days
~
18.7
___.~
100
19.5
20.0
19.1
18.7
~~
Mean diameter of induration in mm
*Transverse diameter of 6 mm or more.
mean diameters of induration obtained to 10 TU of PPD in the Mantoux tests. The mean diameters were all very similar to those resulting from the control lot of vaccine which had been stored at o-5°C. Following this finding, and to establish whether the tuberculin sensitivity was sustained, the school children who had received the vaccine lots which had been stored for 56 days at 0-5°C 22°C and 37°C were tuberculin tested again 14 months after vaccination. The results are summarised in Table IV. The mean diameters of induration were of the same order at 14 months as at 14 weeks post-vaccination. Conversion was 100 per cent from the vaccines which had been stored at 22°C and at 37°C and 96.7 per cent from the vaccine which had been stored at 0-5°C. Discussion Heat stable BCG vaccine is of obvious advantage for successful vaccination in tropical countries. Surface transport without refrigeration is possible and, more important, accidental or unrealised exposure to ambient temperatures for short periods does not destroy the potency of such vaccines. Many years of experience in the manufacture and distribution of vaccine to overseas countries TABLE IV.-SECOND
CLINICAL TEST OF HEAT STABILITY OF BCG DRIED VACCINE BATCH
14
B833 : TUBERCULIN
TESTS AT
MONTHS
--___-~~...~~~~~.._
_
_~____
I
Vaccine stored 56 days at :
o-5°C
/
22°C
37°C
57
39
-__
Number of cases --_ Per cent positive* to 10 TU of PPD at 14 months post-vaccination
61 96.7
Mean diameter of induration in mm *Transverse diameter of 6 mm or more.
100
100
STABILITY
OF BCG
99
have shown that such episodes as storage in unrefrigerated sheds awaiting customs clearance or delayed surface or air transport cannot be entirely avoided. In this paper we have shown that BCG vaccine, made by the process of Ungar and others (1962), will withstand storage at refrigerator temperatures for at least 2 years, temperate room temperature for at least 8 weeks and tropical room temperature (37°C) for 4-8 weeks without an excessive fall in viability. After storage at 37°C for periods up to 56 days, tests on school children showed that the remaining viable organisms were sufficient to achieve a full degree of tuberculin allergy to an intensity similar to that produced by unheated vaccine. Moreover, when the tuberculin tests were done 14 months later, the intensity of the tuberculin allergy was undiminished. Previous experience suggests that this is a good indication of a successful vaccination since, if the effect of the vaccination had been transitory, allergy would start to wane by this time. It therefore seems that vaccine which has been accidentally exposed to elevated temperatures for a few weeks, even in tropical countries, can still be used satisfactorily for vaccination. It is clear, however, that no vaccine will withstand abuse of its recommended storage conditions repeatedly; refrigeration should be used as a routine measure. This is not always realised and it is suggested that caution should be exercised in labelling BCG vaccine ‘heat stable’ in case this leads to carelessness in storing the vaccine. Despite ‘heat stability’ its effective life at elevated temperatures is reduced to weeks rather than years if the recommended refrigerated storage is not adopted. Thanks are due to Mrs A. Stanbrook and Mr J. A. R. Dudley who assisted with the laboratory work, and to Dr J. F. Galloway and Dr G. Ramage for their co-operation in permitting the additional follow-up examinations involved in the clinical evaluation. REFERENCES MILES,A. A., MICRA,S. S. & IRWIN, J. 0. (1938). The estimation
of the bactericidal power of the blood. Journal of
Hvaiene, Cambridpe, 38, 732.
UNGA~~J., MUGGLETO~P. W., DUDLEY,J. A. R. & GRIFFITHS, M. I. (1962). Preparation
dried B.C.G. vaccine of increased stability. British Medical Journal, 2, 1086.
and properties of a freeze-