Heat stability of a recombinant D N A hepatitis B vaccine P . V a n D a m m e $, M . C r a m m * , A. S a f a r y * , P. V a n d e p a p e l i 6 r e * a n d A. M e h e u s *
The heat stability o f a recombinant D N A hepatitis B vaccine was studied in healthy adult volunteers. When compared with vaccine stored at 4°C, heating o f the vaccine for 1 week at 45°C or for 1 month at 37°C did not alter the reactogenicity or the ability o f the vaccine to elicit antibody titres considered to be protective. These results have signifcance in situations where the cold chain is broken, as can happen in countries where proper storage and transport facilities are not always available. Keywords: Heat stability; recombinant DNA; hepatitis B
Hepatitis B is endemic in many countries, especially in Africa and Asia 1. It is a major burden on the health-care systems in those countries, due more to the consequences of chronic hepatitis B virus (HBV)carriage than to acute infection. These facts have considerable importance for the design of vaccination policies 2. No effective and specific therapy is available to treat hepatitis B infection. Therefore, for those living in areas of high endemicity, active immunization to prevent infection is the most efficient way of limiting the spread of hepatitis B. In the countries where vaccination of susceptible people is most necessary, an adequate infrastructure for the distribution of products such as vaccines is essential. The cold chain system is of prime importance because vaccines are sensitive to heat. Even so, refrigerated storage and transport of vaccines may not always be possible and the vaccines can frequently be left at ambient temperatures for extended periods. Heat stability has therefore very important implications on the use of the vaccine and the implementation of a vaccination campaign. This clinical trial was initiated to study the effects on the antibody response elicited following administration of a yeast-derived hepatitis B vaccine (Engerix-B, SmithKline Beecham Biologicals) which had been stored at elevated temperatures. It has been shown that exposure to a temperature of 37°C for 1 week did not alter the immunogenicity of this vaccine 3. 138 healthy adults aged from 18 to 30 years took part in the study. They were randomized into three groups to receive the same lot of yeast-derived vaccine stored in three different ways : the first group received vaccine kept at the normal storage temperature (4°C), the second group received vaccine heated at 45°C for 1 week, and
*University of Antwerp, Department of Epidemiology and Community Medicine, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium. tSmithKline Beecham Biologicals, 89 Rue de I'lnstitut, 1330 Rixensart, Belgium. ~To whom correspondence should be addressed. (Received 23 July 1991; revised 20 November 1991; accepted 28 November 1991) 026Zl-410x/92/060366~)2 ,.(,, 1992 Butterworth-HeinemannLtd 366
Vaccine, Vol. 10, Issue 6, 1992
the third group received vaccine heated at 37°C for 1 month. The three participant groups did not differ with respect to mean ages or sex distribution. Three 20 ~tg vaccine doses were administered intramuscularly in the deltoid region according to a 0, 1, 6 month schedule. Blood was drawn prior to vaccination for measurement of anti-HBc, anti-HBs and HBsAg, and at months 1, 2, 6, 7 and 12 for analysis of anti-HBs. Radioimmunoassay was used to test for the presence of anti-HBs (Ausab, Abbott), HBsAg (Ausria II, Abbott) and anti-HBc (Corab, Abbott ). On the day of vaccination and for the three following days, local and general symptoms were recorded by self-observation on individual symptom sheets. Table 1 shows the antibody response following vaccination with the heat-treated and control vaccines. One month after three vaccine doses 95-100% of subjects had seroconverted, and 6 months after the third vaccination 97-100% of subjects still had measurable titres of anti-HBs. All individuals who responded to the vaccine at month 7 had antibody titres over the generally accepted
T~lble 1 Antibody response following vaccination with either control or heat-treated vaccines GMTs (mlU ml 1) and seroconversion rates
(%) Vaccine
Month 2
Month 7
Month 12
Control
65 45/45 (100)
10 359 33/33 (100)
2018 36/36 (100)
45°C for 1 week
44 40/46 (87)
6813 37/39 (95)
1043 36/37 (97)
37'C for 1 month
48 41/44 (93)
5937 37/37 (100)
1527 36/36 (100)
Antibody response was evaluated using a commercially available radioimmunoassay (Abbott Laboratories). The limit of detection of the assay is 1 mlUml 1; all subjects with undetectable titres were considered to be seronegative. Seroconversion was defined as the induction of anti-HBs titres >1 mlU m1-1 in an initially seronegative subject, Geometric mean anti-HBs titres are shown for seroconverters
Short paper: P. Van Damme et al.
Table 2 Proportion of subjects with protective antibody level (>~10 mlU m1-1) Proportion of subjects (%) seroprotected Vaccine
Month 2
Month 7
Month 12
Control 45°C for 1 week 37°C for 1 month
38/45 (95) 33/46 (72) 33/44 (75)
33/33 (100) 37/39 (95) 37/37 (100)
35/36 (97) 35/37 (95) 35/36 (97)
Table 3 Frequency of solicited local and general symptoms after three vaccine doses
CONCLUSIONS
Vaccine
Symptoms (%)
Control (n = 78)"
45°C for 1 week (n = 86)
37°C for 1 month (n = 84)
Overall data (n = 248)
Local: Soreness Induration
32.1 6.4
31.4 9.3
32.1 11.9
31.9 9.3
29.5 34.6
15.1 23.3 2.3
33.3 34.5 3.6
25.8 30.6 2.0
General: Headache Fatigue Fever
The heat-treated vaccine did not induce a greater frequency, severity or different type of reactions when compared with the 'control' vaccine: 58 % of documented injections in the second group produced symptoms after all three doses compared with 68% and 67% in group 1 and 3, respectively. Globally, soreness at injection site was the most reported local reaction (31.9%) of the documented injections. Fatigue and headache were the most common general reactions, occurring in 30.6% and 25.8% of all reported injections (Table 3). No severe adverse reactions were reported. A similar range of reactions has been reported elsewhere for this vaccine 4"5.
These results show that the reactogenicity and immunogenicity of the vaccine, including its ability to elicit antibody titres considered protective, are not altered by heating for periods of 1 week to 1 month. Thus, although it is recommended to maintain the vaccine at a temperature of between + 2°C and + 8°C for optimal shelf-life and potency, it is reassuring to know that under suboptimal conditions of transport and storage, the vaccine can be expected to maintain immunogenic capacity.
an, number of documented injections
REFERENCES protective level of 10 mlU ml- 1 and 95-97% of subjects were still above this level at month 12 (Table 2). The geometric mean titre ( G M T ) was higher for the control group than for subjects receiving the heated vaccines 1 month after the third dose but the differences were not statistically significant (p = 0.50, by ANOVA one-way test). GMTs at month 12 were still high at 1043-2018 mlU ml- 1 and no statistically significant difference could be shown between the three groups (p = 0.44, by ANOVA one-way test).
1 Prince, AoM Use of hepatitis B vaccine in Africa: rationale and practical approaches for effective utilisation. IARC Sci. Publ. 1984, 63, 337-354 2 Hudson, C.P. How Aids forces reappraisal of hepatitis B virus control in sub-Saharan Africa. Lancet 1990, 336, 1364-1367 3 Just, M. and Berger, R. Immunogenicity of a heat-treated recombinant DNA hepatitis B vaccine. Vaccine 1988, 6, 399-400 4 Wiedermann, G., Scheiermann, N., Goubau, P., Armbrosch, F., Gesermann, M., De Bel, C. et al. Multicentre dose range study of a yeast-derived hepatitis B vaccine. Vaccine 1987, 5, 179 5 Andre, F. and Safary, A. Clinical experience with a yeast-derived hepatitis B vaccine. In: Viral Hepatitis Liver Disease (Ed. Zuckermann, A.), Alan R. Liss., New York, 1988, pp 1025-1030
Vaccine, Vol. 10, Issue 6, 1992
367
The heat stability o f a recombinant D N A hepatitis B vaccine was studied in healthy adult volunteers. When compared with vaccine stored at 4°C, heating o f the vaccine for 1 week at 45°C or for 1 month at 37°C did not alter the reactogenicity or the ability o f the vaccine to elicit antibody titres considered to be protective. These results have signifcance in situations where the cold chain is broken, as can happen in countries where proper storage and transport facilities are not always available. Keywords: Heat stability; recombinant DNA; hepatitis B
Hepatitis B is endemic in many countries, especially in Africa and Asia 1. It is a major burden on the health-care systems in those countries, due more to the consequences of chronic hepatitis B virus (HBV)carriage than to acute infection. These facts have considerable importance for the design of vaccination policies 2. No effective and specific therapy is available to treat hepatitis B infection. Therefore, for those living in areas of high endemicity, active immunization to prevent infection is the most efficient way of limiting the spread of hepatitis B. In the countries where vaccination of susceptible people is most necessary, an adequate infrastructure for the distribution of products such as vaccines is essential. The cold chain system is of prime importance because vaccines are sensitive to heat. Even so, refrigerated storage and transport of vaccines may not always be possible and the vaccines can frequently be left at ambient temperatures for extended periods. Heat stability has therefore very important implications on the use of the vaccine and the implementation of a vaccination campaign. This clinical trial was initiated to study the effects on the antibody response elicited following administration of a yeast-derived hepatitis B vaccine (Engerix-B, SmithKline Beecham Biologicals) which had been stored at elevated temperatures. It has been shown that exposure to a temperature of 37°C for 1 week did not alter the immunogenicity of this vaccine 3. 138 healthy adults aged from 18 to 30 years took part in the study. They were randomized into three groups to receive the same lot of yeast-derived vaccine stored in three different ways : the first group received vaccine kept at the normal storage temperature (4°C), the second group received vaccine heated at 45°C for 1 week, and
*University of Antwerp, Department of Epidemiology and Community Medicine, Universiteitsplein 1, 2610 Wilrijk, Antwerp, Belgium. tSmithKline Beecham Biologicals, 89 Rue de I'lnstitut, 1330 Rixensart, Belgium. ~To whom correspondence should be addressed. (Received 23 July 1991; revised 20 November 1991; accepted 28 November 1991) 026Zl-410x/92/060366~)2 ,.(,, 1992 Butterworth-HeinemannLtd 366
Vaccine, Vol. 10, Issue 6, 1992
the third group received vaccine heated at 37°C for 1 month. The three participant groups did not differ with respect to mean ages or sex distribution. Three 20 ~tg vaccine doses were administered intramuscularly in the deltoid region according to a 0, 1, 6 month schedule. Blood was drawn prior to vaccination for measurement of anti-HBc, anti-HBs and HBsAg, and at months 1, 2, 6, 7 and 12 for analysis of anti-HBs. Radioimmunoassay was used to test for the presence of anti-HBs (Ausab, Abbott), HBsAg (Ausria II, Abbott) and anti-HBc (Corab, Abbott ). On the day of vaccination and for the three following days, local and general symptoms were recorded by self-observation on individual symptom sheets. Table 1 shows the antibody response following vaccination with the heat-treated and control vaccines. One month after three vaccine doses 95-100% of subjects had seroconverted, and 6 months after the third vaccination 97-100% of subjects still had measurable titres of anti-HBs. All individuals who responded to the vaccine at month 7 had antibody titres over the generally accepted
T~lble 1 Antibody response following vaccination with either control or heat-treated vaccines GMTs (mlU ml 1) and seroconversion rates
(%) Vaccine
Month 2
Month 7
Month 12
Control
65 45/45 (100)
10 359 33/33 (100)
2018 36/36 (100)
45°C for 1 week
44 40/46 (87)
6813 37/39 (95)
1043 36/37 (97)
37'C for 1 month
48 41/44 (93)
5937 37/37 (100)
1527 36/36 (100)
Antibody response was evaluated using a commercially available radioimmunoassay (Abbott Laboratories). The limit of detection of the assay is 1 mlUml 1; all subjects with undetectable titres were considered to be seronegative. Seroconversion was defined as the induction of anti-HBs titres >1 mlU m1-1 in an initially seronegative subject, Geometric mean anti-HBs titres are shown for seroconverters
Short paper: P. Van Damme et al.
Table 2 Proportion of subjects with protective antibody level (>~10 mlU m1-1) Proportion of subjects (%) seroprotected Vaccine
Month 2
Month 7
Month 12
Control 45°C for 1 week 37°C for 1 month
38/45 (95) 33/46 (72) 33/44 (75)
33/33 (100) 37/39 (95) 37/37 (100)
35/36 (97) 35/37 (95) 35/36 (97)
Table 3 Frequency of solicited local and general symptoms after three vaccine doses
CONCLUSIONS
Vaccine
Symptoms (%)
Control (n = 78)"
45°C for 1 week (n = 86)
37°C for 1 month (n = 84)
Overall data (n = 248)
Local: Soreness Induration
32.1 6.4
31.4 9.3
32.1 11.9
31.9 9.3
29.5 34.6
15.1 23.3 2.3
33.3 34.5 3.6
25.8 30.6 2.0
General: Headache Fatigue Fever
The heat-treated vaccine did not induce a greater frequency, severity or different type of reactions when compared with the 'control' vaccine: 58 % of documented injections in the second group produced symptoms after all three doses compared with 68% and 67% in group 1 and 3, respectively. Globally, soreness at injection site was the most reported local reaction (31.9%) of the documented injections. Fatigue and headache were the most common general reactions, occurring in 30.6% and 25.8% of all reported injections (Table 3). No severe adverse reactions were reported. A similar range of reactions has been reported elsewhere for this vaccine 4"5.
These results show that the reactogenicity and immunogenicity of the vaccine, including its ability to elicit antibody titres considered protective, are not altered by heating for periods of 1 week to 1 month. Thus, although it is recommended to maintain the vaccine at a temperature of between + 2°C and + 8°C for optimal shelf-life and potency, it is reassuring to know that under suboptimal conditions of transport and storage, the vaccine can be expected to maintain immunogenic capacity.
an, number of documented injections
REFERENCES protective level of 10 mlU ml- 1 and 95-97% of subjects were still above this level at month 12 (Table 2). The geometric mean titre ( G M T ) was higher for the control group than for subjects receiving the heated vaccines 1 month after the third dose but the differences were not statistically significant (p = 0.50, by ANOVA one-way test). GMTs at month 12 were still high at 1043-2018 mlU ml- 1 and no statistically significant difference could be shown between the three groups (p = 0.44, by ANOVA one-way test).
1 Prince, AoM Use of hepatitis B vaccine in Africa: rationale and practical approaches for effective utilisation. IARC Sci. Publ. 1984, 63, 337-354 2 Hudson, C.P. How Aids forces reappraisal of hepatitis B virus control in sub-Saharan Africa. Lancet 1990, 336, 1364-1367 3 Just, M. and Berger, R. Immunogenicity of a heat-treated recombinant DNA hepatitis B vaccine. Vaccine 1988, 6, 399-400 4 Wiedermann, G., Scheiermann, N., Goubau, P., Armbrosch, F., Gesermann, M., De Bel, C. et al. Multicentre dose range study of a yeast-derived hepatitis B vaccine. Vaccine 1987, 5, 179 5 Andre, F. and Safary, A. Clinical experience with a yeast-derived hepatitis B vaccine. In: Viral Hepatitis Liver Disease (Ed. Zuckermann, A.), Alan R. Liss., New York, 1988, pp 1025-1030
Vaccine, Vol. 10, Issue 6, 1992
367
