- Email: [email protected]
Immunogenicity of recombinant hepatitis B vaccine in dialysis patients
190
Journal of Hepatology, 1986;3:190-195
Elsevier HEP 00204
Immunogenicity of Recombinant Hepatitis B Vaccine in Dialysis Patients
W. Jilg, M. Schmidt, B. Weinel, Th. Kfittler, H. Brass, J. Bommer, R. M/Jller, B. Schulte, A. Schwarzbeck and F. Deinhardt Mat" von Pettenkofer Institute for Hygiene and Medical Microbiology, University of Munich, Munich; City Hospitals, Ludwigshafen am Rh ein; Department of Medicine, University of Heidelberg, Heidelberg; School of Medicine, Hanover; and Hospital for Kidney and High Blood Pressure Diseases, Hanover (F. R. G.)
(Received 31 January, 1986) (Accepted 8 May, 1986)
Summary Eighty-eight dialysis patients were vaccinated with recombinant hepatitis B vaccine prepared in yeast. Fourtynine patients were immunized 3 times (months 0, 1, 6) intragluteally with 40 pg hepatitis B surface antigen (HBsAg) per dose. Only 32 of them (65.3%) showed anti-HBs concentrations above 10 IU/i with a geometric mean titer (GMT) of 180.7 IU/I after 3 vaccinations, whereas all of the 16 healthy controls, vaccinated 3 times with a 10-ktg dose of the same vaccine batch, had specific antibodies higher than 10 IU/! (GMT 897.4 IU/I). Responses of patients were slightly higher than those of dialysis patients vaccinated in an earlier study with plasmaderived vaccine according to the same schedule. Results in 20 patients immunized 6 times intragluteally with 40 pg HBsAg/dose in monthly intervals were not better (at month 7, 65% showed anti-HBs concentrations > 10 IU/I; GMT = 126.6 IU/1), and 19 patients receiving 6 times 20 pg HBsAg monthly showed significantly lower responses (anti-HBs > 10 IU/I in 42% of vaccinees, GMT = 89.5 IU/I). The vaccine was tolerated well; side-effects were slight, and no serious adverse reactions were observed. In conclusion, recombinant hepatitis B vaccine is comparable to plasma-derived vaccine also in the case of dialysis patients; a 6-dose schedule does not seem to have much advantage compared to the conventional 3-dose regimen.
Introduction At present, vaccination against hepatitis B is the most important measure in the prevention of this dis-
ease; the efficacy and safety of currently available vaccines have been demonstrated in numerous studies [1]. However, as production of vaccines from the plasma of chronic carriers of hepatitis B surface anti-
Correspondenceshould be addressed to Dr. WolfgangJilg, Pettenkofer Institute, Pettenkoferstrasse 9a, D-8000Munich 2, F.R.G., Tel. (089)539321. 0168-8278/86/$03.50© 1986Elsevier SciencePublishers B.V. (BiomedicalDivision)
191
RECOMBINANT HEPATITIS B VACCINE gen (HBsAg) is difficult and expensive, alternative sources have been sought [2]. Recently, the first clinical trials of hepatitis B vaccines made from recombinant yeast showed good immunogenicity and proved to be safe in healthy adults [3-9]. Dialysis patients are at high risk from hepatitis B, but their impaired immune systems lead to poor responses to vaccination with plasma-derived hepatitis B vaccine [10-18]. The following study evaluated the immune response of dialysis patients to a recombinant vaccine. The immune responses of 88 dialysis patients to this vaccine are compared to those of healthy adults, and to those of a similar patient group vaccinated earlier with plasma-derived vaccine [13-15].
Patients and Methods
The 88 patients came from 3 German haemodialysis centres. In all centres, the incidence of hepatitis B was low (no new hepatitis B cases during the previous 3 years). In one of the centres, 58 patients were distributed randomly in 3 groups (Ia, II, III) and immunized according to 3 different vaccination schedules (see below). Patients of the two other centres togeth-
er with group Ia patients were enrolled in group I. Sixteen healthy controls were recruited from medical and laboratory personnel. The 75 patients in the plasma vaccine group came from earlier studies in one of the above centres [13] and from 4 dialysis units in Munich [16]. The age and sex distribution of the different groups are shown in Table 1. No statistically significant differences were found between the mean ages of different patient groups (groups I - I I I , plasma vaccine group); healthy controls, however, were significantly younger. Before vaccination, all subjects were negative for H B s A g , antibodies against H B s A g (anti-HBs) and antibodies against hepatitis B core antigen (anti-HBc), and their aminotransferase levels were normal (alanine and aspartate aminotransferase ~< 17 IU/I and ~< 19 IU/I, respectively). All participants in the study kept daily records of body temperature and side-effects for 5 days after each injection. The recombinant hepatitis B vaccine (lot 986/CK 733, containing 20/~g of HBsAg/ml) was prepared by Merck, Sharp & D o h m e Research Laboratories. Dialysis patients of group I received 3 doses with 40/~g of H B s A g (2 ml of vaccine) at 0, 1 and 6 months. Participants in groups II and III were injected 6 times in
TABLE 1 SEX AND AGE DISTRIBUTION OF PARTICIPANTS Total n/mean age (yr)* + S.D.
Female n/mean age (yr) + S.D.
Male n/mean age (yr) + S.D.
Dialysis patients (recomb. vaccine) Group I Group Iaa Group II Group III
49/52.5 + 19/55.8 + 19/60.5 + 20/59.4 +
22/53.5 + 10/51.5 + 11/57.7 + 10/64.6 +
27/51.6 + 9/61.0 + 8/63.4 + 10/54.1 +
Healthy controls (recomb. vaccine)
16/29.3 + 10.4
9/26.4 + 12.5
7/33.5 + 8.8
Dialysis patients b (plasma vaccine)
75/47.0 + 14.1
33/50.1 + 12.0
42/44.5 + 15.1
12.9 15.2 13.0 12.6
12.2 14.9 14.8 11.0
13.3 13.9 10.4 11.1
* Statistical significant differences were found between the healthy control group and group I (P < 0.01). None of the differences between group I and the plasma vaccine group, and groups Ia, II and III were statistically significant (P > 0.05, Wilcoxon's rank-sum test). Subgroup of group I. b Vaccinated in an earlier study.
192 monthly intervals (months 0-5): group II patients received single doses of 20/~g HBsAg; patients in group III received doses of 40/~g HBsAg at each injection. All patients were injected intragluteally. Participants of the control group received 0.5 ml of vaccine (10 ~g HBsAg) at months 0, 1 and 6; vaccine was administered intramuscularly in the upper arm. The 75 patients vaccinated earlier with plasma-derived vaccine were injected 3 times (months 0, 1, 6) intragluteally with 40~g HBsAg/dose. Blood samples were taken on the day of the first vaccination and then monthly. HBsAg, anti-HBs and anti-HBc were tested by radioimmunoassay with commercially available test kits ('AuSRIA II', 'AuSAB', 'CORAB', Abbott Laboratories, North Chicago). Anti-HBs concentrations were calculated using a standard curve ranging from 5 to 150 IU/I; the standard serum was calibrated with the first W H O reference preparation from 1977 [19]. Seroconversion was assumed to have occurred when anti-HBs in one serum sample became positive according to the criteria for the AUSAB-test (ratio of counts/min of sample to counts/min of negative control 2.1), and other reasons for seroconversion (blood transfusion or immunoglobulin injection) could be excluded. Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were determined using commercially available kits (monotest GPT, monotest GOT, Boehringer, Mannheim). Overall immune responses (anti-HBs concentrations of all participants) were compared using Wilcoxon's rank-sum test. The chi-square-test was used for comparison of percentages (seroconversion rates, rates of subjects with anti-HBs > 10 IU/1).
Results
lmmunogenicity of triple vaccination with 40 i~g HBsAg Forty-nine patients received 3 doses of 40 ~g at months 0, 1 and 6. The 16 control group participants were vaccinated by the same schedule but with only 10 ~g HBsAg/dose. Immune responses were followed by monthly anti-HBs determinations. At all
w. JILG et al. ANTI-HBS lUlL , H
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Fig. 1. Anti-HBs responses (GMTs, all subjects) of dialysis pa-
tients (n = 49) and healthy controls (n = 16) after vaccination with recombinant hepatitis B vaccine and of dialysis patients vaccinated in an earlier study with plasma derived vaccine (n = 75).
times tested, seroconversion rates and geometric mean anti-HBs titers (GMT) were significantly lower in the patient than in the control group (Fig. 1). Four weeks after the third injection, 34 patients (69.4%) had antibodies against HBsAg; 32 (65.3%) had antiHBs levels above 10 IU/I. The G M T of all vaccinees .was 36.8 IU/I. All healthy controls were anti-HBspositive at that time with values > 100 IU/I and a G M T of 897.4 IU/I (Table 2). The stud); group was compared retroactively with a control group of dialysis patients with a similar sex and age distribution which was vaccinated 3 times with 40 ~g HBsAg of plasma-derived vaccine in an earlier study [13,16]. During the first 5 months, immune responses were comparable in both groups; at 7 months, patients vaccinated with recombinant vaccine showed a slightly higher seroconversion rate and G M T and a slightly but significantly higher proportion of vaccinees with anti-HBs concentrations above 10 IU/I (Fig. 1, Table 2). Female patients showed a slightly better immune response to recombinant vaccine than male patients: 78% of women were anti-HBs-positive at month 7, with a G M T of 224 IU/I, as compared to 62% and 142 IU/I in men. These differences, however, were not statistically significant.
RECOMBINANT HEPATITIS B VACCINE
193
TABLE 2 ANTI-HBS ONE MONTH AFTER THE THIRD VACCINATION (MONTH 7)
Mean age ( y r ) ± S D Anti-HBs-positive > 10 IU/I > 100 IU/I > 1000 IU/I > 10000 IU/I
Dialysis patients (recombinant vaccine) total(n = 49)
Healthy controls (recombinant vaccine) (n = 16)
Dialysis patientsa (plasmavaccine) (n = 75)
52.5 ± 12.9
29.3 ± 10.4
47.0±14.1
100.0% 100.0% 100.0% 62.5% 0
52.0% 42.7%* 29.4% 4.1% 0
897.4 IU/I 897.4 IU/I
10.4 IU/I 89.8 IU/I
69.4% 65.3%* 34.7% 16.3% 2.0%
Geometric mean anti-HBs all: responders:
36.8 IU/I 180.7 IU/I
" Vaccinated in an earlier study. * P = 0.014 (chi-square test).
Comparison of 6-dose and 3-dose regimens Thirty-nine patients were vaccinated 6 times at monthly intervals (months 0 - 5 ) with either 20/~g H B s A g (group II, n = 19) or 40 p g H B s A g (group III, n = 20), and the results were c o m p a r e d to those of 20 patients with similar sex and age distribution vaccinated in the same dialysis unit by a 3-dose regimen (group Ia, subgroup of group I). D u r i n g the first 6 months, all 3 groups showed a steady increase in G M T and seroconversion rates with significant differences between the 3 groups (Figs. 2 and 3) at month 6: group III showed the highest seroconver-
sion rate (72%) and G M T (69 IU/I) followed by group II (6 x 20/~g) (53% and 10.0 IU/I) and Ia (3 x 40/~g) (37% and 3.4 IU/I) ( I a v s II: P < 0.05; I a v s III: P < 0.01; II vs III: P < 0.05). 26%, 37% and 70% of vaccinees, respectively, had anti-HBs levels greater than 10 IU/1 in groups Ia, II and III at this time. H o w e v e r , the third injection in group Ia at 6 months resulted in a m a r k e d increase in seroconversion (63%) and G M T (23 IU/1) as m e a s u r e d one month later, whereas at this time the mean G M T in group II was essentially unchanged (10.8 IU/I), and had already decreased in group III (35.4 IU/I). The ANTI-HBS IU/Lt
SEROCONVERSION %, 100
1000 "1
/
I~1 GROUP l a , 3 x 40 tJg HI]SAG r ~ l GROUP 2. 6 • 20 pg
HGSAg
GROUP 3. 6 • 40 pg HBsAG
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GROUP 1 a , 3 x 40pg HGsAg
~
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=--A
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10~
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75 50 25 N
!
1
• •
:3
4
,5
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•
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6
7
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Fig. 2. Seroconversion rates of dialysis patients after vaccination with 3 x 40 lzg HBsAg (group Ia, n = 19), 6 x 20/~g
HBsAg (group 11, n = 19) or 6 x 40pg HBsAg (group ill, n = 20) of recombinant hepatitis B vaccine.
i •
•
•
•
2
5
4
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6 •
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•
•
•
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V A C C I N A T I O NGROUPS S 2 ANO 3
Fig. 3. Anti-HBs responses (GMTs, all subjects) of dialysis patients after vaccination with 3 x 40/tg HBsAg (group Ia, n = 19), 6 x 20/~g HBsAg (group II, n = 19) or 6 x 40Fg HBsAg (group III, n = 20) of recombinant hepatitis B vaccine.
194 percentages of vaccinees with anti-HBs values > 10 IU were now 55%, 42% and 74% in groups Ia, II and III. Differences in GMT, between groups Ia and III were no longer significant (P > 0.10); in fact, 3 months later group Ia showed the highest GMT. Seroconversion rates and percentages of vaccinees with anti-HBs > 10 IU were now 63% and 53% for group Ia, 53% and 53% for group II and 74% and 61% for group III.
Side-effects In general, the vaccine was tolerated very well. Besides minor local symptoms such as transient pain, burning, itching and slight swelling at the injection site or systemic symptoms including headache, nausea or dizziness, which were reported after 23 of 393 injections, no serious adverse reactions were observed. In most patients, body temperatures did not rise significantly within 5 days after each vaccination. Temperatures of 37.5 or higher were measured in 24 patients; 4 patients had fevers between 38.0 and 38.8. There was no difference in severity and frequency of symptoms in patients vaccinated 3 times or 6 times, or in patients and healthy controls.
Discussion
The aim of this study was the evaluation of a new, recombinant hepatitis B vaccine in dialysis patients. In most trials, the immune response of these patients to plasma-derived hepatitis B vaccines has been poor, which is probably due to chronic uraemia rather than to dialysis as similar results were observed in uraemic patients vaccinated before dialysis [12,13]; the cause underlying the poor response to such hepatitis B vaccines is unknown. Similarly, in our study, the seroconversion rates and G M T of 49 dialysis patients vaccinated 3 times with recombinant hepatitis B vaccine containing 40 pg HBsAg per dose were considerably lower than the values of a control group of healthy adults vaccinated with the same batch of vaccine but with 10 pg HBsAg per single dose. As there were considerable differences in mean ages between these two groups, a di-
w. JILG et al. rect comparison was not possible; however, results in our control group were similar to those obtained in healthy persons with other batches of recombinant vaccine [4] or with plasma-derived vaccine [22] demonstrating good immunogenicity of the vaccine used in the present study. The immune response of the patients was comparable to that of dialysis patients vaccinated earlier with 40 /~g/dose of plasma-derived vaccine. The proportion of patients with anti-HBs concentrations of > 10 IU/! was slightly, but significantly higher after recombinant vaccine; however, in a historical comparison, relatively small differences such as this one should not be overinterpreted. A comparison with other vaccination studies with plasma-derived vaccine carried out in patient groups of similar size and composition, shows that results obtained in our study group were indeed similar to the majority of trials, in which 53-75% of patients had anti-HBs values of more than 10 IU/I with GMTs of 85-232 IU/I [10-12,15,18]. Only two studies reported clearly higher values: the study of Benhamou et al. [17] (82% seroconversion, G M T 1123 IU/I), and the study of Desmyter et al. [20] using the semipurified and only heat-inactivated Netherlands Red Cross vaccine (90% seroconversion, G M T 1586 I.U/1). The influence of sex on the immune response was demonstrated by a slight trend to higher seroconversion rates and anti-HBs concentrations in women. However, these differences were neither statistically significant nor as pronounced as differences found in healthy subjects [21,24]. Also, as shown by other studies [11,20], the immune impairment due to chronic uraemia seems to be the overriding influence on the immune response of these patients. Since vaccination schedules using more frequent injections have been successful [13,14,17] we studied the efficiency of a multi-dose regimen in 39 patients using 6 monthly injections of either 20 or 40 pg of vaccine. However, according to our results the benefits of this schedule are doubtful because after 7 and 10 months no statistically significant differences in seroconversion and GMTs could be found in patients injected 6 times with 40 pg HBsAg compared to those vaccinated only 3 times with 40 pg; an even signifi-
195
RECOMBINANT HEPATITIS B VACCINE cantly lower G M T was seen in the 6 times 20-pg group. A similar observation was reported by Benhamou et al. [17]: a better response was noted in dialysis patients after 3 injections of a double dose than after 4 injections of the standard dose. In conclusion, although a comparison with the other studies is difficult due to the heterogeneity of age distribution, time on dialysis and underlying disease of patient groups, our results indicate that in dialysis patients r e c o m b i n a n t hepatitis B vaccine seems to be as immunogenic as the two licensed plasma-derived vaccines. Side-effects were negligible, not differing from those seen after plasma vaccine. Thus, recombinant vaccines can replace plasma-derived vaccines
References 1 Stevens CE, Taylor PE, Tong MJ, et al. Hepatitis B vaccine - - An overview. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease, Grune and Stratton, Orlando, 1984: 275-291. 2 Purcell RH, Gerin JL. Prospects for second and third generation hepatitis B vaccines. Hepatology 1985; 5: 159-163. 3 Scolnick EM, McLean AA, West DJ, et al. Clinical evaluation in healthy adults of a hepatitis B vaccine made by recombinant DNA. J Amer Med Ass 1984; 251: 2812-2815. 4 Jilg W, Lorbeer B, Schmidt M, et al. Clinical evaluation of a recombinant hepatitis B vaccine. Lancet i984; ii: 1174-1175. 5 Davidson M, Krugman S. Immunogenicity of recombinant yeast hepatitis B vaccine. Lancet 1985; i: 108-109. 6 Dandolos E, Roumeliotou-Karayannis A, Richardson SC, Papaevangelou G. Safety and immunogenicity of a recombinant hepatitis B vaccine. J Med Virol 1985; 17: 57-62. 7 Hollinger FB, Troisi CL. Anti-HBs responses to vaccination with a human hepatitis B vaccine made by recombinant DNA technology in yeast. J Infect Dis 1985; 153: 156-159. 8 Heijtink RA, Kruining J, Bakker M, Schalm SW. Immune response after vaccination with recombinant hepatitis B vaccine as compared to that after plasma-derived vaccine. Antiviral Res 1985; Suppl. I: 273-279. 9 Kuwert E, Scheiermann N, Gesemann M, et al. Dose range study in healthy volunteers of a hepatitis B vaccine produced in yeast. Antiviral Res 1985; Suppl. I: 281-288. 10 Crosnier J, Jungers P, Courouce AM, et al. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in french haemodialysis units: II, Haemodialysis patients. Lancet 1981; i: 797. 11 Goudeau A, Coursaget P, Barin F, et al. Prevention of hepatitis B by active and passive-active immunization. In: W Szmuness, HJ Alter, JE Maynard (Eds.), Viral Hepatitis. Franklin Institute Press, Philadelphia, PA, 1982: 509-525.
for dialysis patients. Nevertheless, an improvement of the i m m u n e response of these patients is needed, perhaps by increasing single doses, by the use of immuno-stimulators, by the use of more immunogenic vaccine preparations such as micelle vaccines [22] or vaccines containing the pre-S sequence of HBsAg [2].
Acknowledgements We want to thank Liane Sakreida for technical assistance and Dr. Jean B. Deinhardt for her help in preparing the manuscript.
12 Grob PJ, Binswanger U, Zaruba K, et al. Immunogenicity of a hepatitis B subunit vaccine in hemodialysis and in renal transplant recipients. Antiviral Res 1983;3: 43-52. 13 Bommer J, Deinhardt F, Jilg W, et al. Impfung ur~imischer Patienten gegen Hepatitis B, Dtsch Med Wschr 1983; 108: 1823-1826. 14 Bommer J, Grussendorf M, Jilg W, et al. Vaccination against hepatitis B in patients with renal insufficiency, Proc EDTA-ERA 1984; 21: 300-305. 15 Miiller R, Arnold W, Deinhardt F, et al. Hepatitis B Impfung von immunsupprimierten Patienten. In: F Deinhardt and H Spiess (Eds.), Impfung gegen Hepatitis B. Die Medizinische Verlagsgesellschaft, Marburg/Lahn, 1983: 51-54. 16 Jilg W, Zachoval R, Schmidt M, Deinhardt F. Aktive und passive-aktive Impfung gegen Hepatitis B. In: F Deinhardt and H Spiess (Eds.), Impfung gegen Hepatitis B. Die Medizinische Verlagsgesellschaft, Marburg/Lahn,, 1983: 39-50. 17 Benhamou E, Courouce AM, Jungen P, et al. Hepatitis B vaccine: randomised trial of immunogenicity in haemodialysis patients. Clin Nephrol 1984;21: 143-147. 18 Stevens CE, Alter HJ, Taylor PE, et al. Hepatitis B vaccine in patients receiving hemodialysis, N Engl J Med 1984; 311: 496-501. 19 World Health Organization, WHO Technical Report Series No. 638. WHO, Geneva, 1979: 29. 20 Desmyter J, Colaert J, Comparative immunogenicity of MSD, Pasteur and CLB hepatitis B vaccines in 245 hemodialysis patients. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stranon, Orlando, 1984: 709-710. 21 Zachoval R, Jilg W, Lorbeer B, et al. Passive/active immunization against hepatitis B. J Infect Dis 1984; 150: 112-117. 22 Howard CR, Skelly J, Tsiquaye KN, et al. The development and properties of alternative hepatitis B polypeptide vaccines. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stratton, Orlando, 1984: 410-423.
Journal of Hepatology, 1986;3:190-195
Elsevier HEP 00204
Immunogenicity of Recombinant Hepatitis B Vaccine in Dialysis Patients
W. Jilg, M. Schmidt, B. Weinel, Th. Kfittler, H. Brass, J. Bommer, R. M/Jller, B. Schulte, A. Schwarzbeck and F. Deinhardt Mat" von Pettenkofer Institute for Hygiene and Medical Microbiology, University of Munich, Munich; City Hospitals, Ludwigshafen am Rh ein; Department of Medicine, University of Heidelberg, Heidelberg; School of Medicine, Hanover; and Hospital for Kidney and High Blood Pressure Diseases, Hanover (F. R. G.)
(Received 31 January, 1986) (Accepted 8 May, 1986)
Summary Eighty-eight dialysis patients were vaccinated with recombinant hepatitis B vaccine prepared in yeast. Fourtynine patients were immunized 3 times (months 0, 1, 6) intragluteally with 40 pg hepatitis B surface antigen (HBsAg) per dose. Only 32 of them (65.3%) showed anti-HBs concentrations above 10 IU/i with a geometric mean titer (GMT) of 180.7 IU/I after 3 vaccinations, whereas all of the 16 healthy controls, vaccinated 3 times with a 10-ktg dose of the same vaccine batch, had specific antibodies higher than 10 IU/! (GMT 897.4 IU/I). Responses of patients were slightly higher than those of dialysis patients vaccinated in an earlier study with plasmaderived vaccine according to the same schedule. Results in 20 patients immunized 6 times intragluteally with 40 pg HBsAg/dose in monthly intervals were not better (at month 7, 65% showed anti-HBs concentrations > 10 IU/I; GMT = 126.6 IU/1), and 19 patients receiving 6 times 20 pg HBsAg monthly showed significantly lower responses (anti-HBs > 10 IU/I in 42% of vaccinees, GMT = 89.5 IU/I). The vaccine was tolerated well; side-effects were slight, and no serious adverse reactions were observed. In conclusion, recombinant hepatitis B vaccine is comparable to plasma-derived vaccine also in the case of dialysis patients; a 6-dose schedule does not seem to have much advantage compared to the conventional 3-dose regimen.
Introduction At present, vaccination against hepatitis B is the most important measure in the prevention of this dis-
ease; the efficacy and safety of currently available vaccines have been demonstrated in numerous studies [1]. However, as production of vaccines from the plasma of chronic carriers of hepatitis B surface anti-
Correspondenceshould be addressed to Dr. WolfgangJilg, Pettenkofer Institute, Pettenkoferstrasse 9a, D-8000Munich 2, F.R.G., Tel. (089)539321. 0168-8278/86/$03.50© 1986Elsevier SciencePublishers B.V. (BiomedicalDivision)
191
RECOMBINANT HEPATITIS B VACCINE gen (HBsAg) is difficult and expensive, alternative sources have been sought [2]. Recently, the first clinical trials of hepatitis B vaccines made from recombinant yeast showed good immunogenicity and proved to be safe in healthy adults [3-9]. Dialysis patients are at high risk from hepatitis B, but their impaired immune systems lead to poor responses to vaccination with plasma-derived hepatitis B vaccine [10-18]. The following study evaluated the immune response of dialysis patients to a recombinant vaccine. The immune responses of 88 dialysis patients to this vaccine are compared to those of healthy adults, and to those of a similar patient group vaccinated earlier with plasma-derived vaccine [13-15].
Patients and Methods
The 88 patients came from 3 German haemodialysis centres. In all centres, the incidence of hepatitis B was low (no new hepatitis B cases during the previous 3 years). In one of the centres, 58 patients were distributed randomly in 3 groups (Ia, II, III) and immunized according to 3 different vaccination schedules (see below). Patients of the two other centres togeth-
er with group Ia patients were enrolled in group I. Sixteen healthy controls were recruited from medical and laboratory personnel. The 75 patients in the plasma vaccine group came from earlier studies in one of the above centres [13] and from 4 dialysis units in Munich [16]. The age and sex distribution of the different groups are shown in Table 1. No statistically significant differences were found between the mean ages of different patient groups (groups I - I I I , plasma vaccine group); healthy controls, however, were significantly younger. Before vaccination, all subjects were negative for H B s A g , antibodies against H B s A g (anti-HBs) and antibodies against hepatitis B core antigen (anti-HBc), and their aminotransferase levels were normal (alanine and aspartate aminotransferase ~< 17 IU/I and ~< 19 IU/I, respectively). All participants in the study kept daily records of body temperature and side-effects for 5 days after each injection. The recombinant hepatitis B vaccine (lot 986/CK 733, containing 20/~g of HBsAg/ml) was prepared by Merck, Sharp & D o h m e Research Laboratories. Dialysis patients of group I received 3 doses with 40/~g of H B s A g (2 ml of vaccine) at 0, 1 and 6 months. Participants in groups II and III were injected 6 times in
TABLE 1 SEX AND AGE DISTRIBUTION OF PARTICIPANTS Total n/mean age (yr)* + S.D.
Female n/mean age (yr) + S.D.
Male n/mean age (yr) + S.D.
Dialysis patients (recomb. vaccine) Group I Group Iaa Group II Group III
49/52.5 + 19/55.8 + 19/60.5 + 20/59.4 +
22/53.5 + 10/51.5 + 11/57.7 + 10/64.6 +
27/51.6 + 9/61.0 + 8/63.4 + 10/54.1 +
Healthy controls (recomb. vaccine)
16/29.3 + 10.4
9/26.4 + 12.5
7/33.5 + 8.8
Dialysis patients b (plasma vaccine)
75/47.0 + 14.1
33/50.1 + 12.0
42/44.5 + 15.1
12.9 15.2 13.0 12.6
12.2 14.9 14.8 11.0
13.3 13.9 10.4 11.1
* Statistical significant differences were found between the healthy control group and group I (P < 0.01). None of the differences between group I and the plasma vaccine group, and groups Ia, II and III were statistically significant (P > 0.05, Wilcoxon's rank-sum test). Subgroup of group I. b Vaccinated in an earlier study.
192 monthly intervals (months 0-5): group II patients received single doses of 20/~g HBsAg; patients in group III received doses of 40/~g HBsAg at each injection. All patients were injected intragluteally. Participants of the control group received 0.5 ml of vaccine (10 ~g HBsAg) at months 0, 1 and 6; vaccine was administered intramuscularly in the upper arm. The 75 patients vaccinated earlier with plasma-derived vaccine were injected 3 times (months 0, 1, 6) intragluteally with 40~g HBsAg/dose. Blood samples were taken on the day of the first vaccination and then monthly. HBsAg, anti-HBs and anti-HBc were tested by radioimmunoassay with commercially available test kits ('AuSRIA II', 'AuSAB', 'CORAB', Abbott Laboratories, North Chicago). Anti-HBs concentrations were calculated using a standard curve ranging from 5 to 150 IU/I; the standard serum was calibrated with the first W H O reference preparation from 1977 [19]. Seroconversion was assumed to have occurred when anti-HBs in one serum sample became positive according to the criteria for the AUSAB-test (ratio of counts/min of sample to counts/min of negative control 2.1), and other reasons for seroconversion (blood transfusion or immunoglobulin injection) could be excluded. Serum alanine aminotransferase (ALT) and serum aspartate aminotransferase (AST) were determined using commercially available kits (monotest GPT, monotest GOT, Boehringer, Mannheim). Overall immune responses (anti-HBs concentrations of all participants) were compared using Wilcoxon's rank-sum test. The chi-square-test was used for comparison of percentages (seroconversion rates, rates of subjects with anti-HBs > 10 IU/1).
Results
lmmunogenicity of triple vaccination with 40 i~g HBsAg Forty-nine patients received 3 doses of 40 ~g at months 0, 1 and 6. The 16 control group participants were vaccinated by the same schedule but with only 10 ~g HBsAg/dose. Immune responses were followed by monthly anti-HBs determinations. At all
w. JILG et al. ANTI-HBS lUlL , H
PATIENTS,RECOMB VACC,
I1---.11 PATIENTS. PLASMA VACC
I000 "
~lb~,e
CONTROLS, RECOMB VACC
100-
10-
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i •
•
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3
4
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6 •
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1'2 MONT~ VACCINATION
Fig. 1. Anti-HBs responses (GMTs, all subjects) of dialysis pa-
tients (n = 49) and healthy controls (n = 16) after vaccination with recombinant hepatitis B vaccine and of dialysis patients vaccinated in an earlier study with plasma derived vaccine (n = 75).
times tested, seroconversion rates and geometric mean anti-HBs titers (GMT) were significantly lower in the patient than in the control group (Fig. 1). Four weeks after the third injection, 34 patients (69.4%) had antibodies against HBsAg; 32 (65.3%) had antiHBs levels above 10 IU/I. The G M T of all vaccinees .was 36.8 IU/I. All healthy controls were anti-HBspositive at that time with values > 100 IU/I and a G M T of 897.4 IU/I (Table 2). The stud); group was compared retroactively with a control group of dialysis patients with a similar sex and age distribution which was vaccinated 3 times with 40 ~g HBsAg of plasma-derived vaccine in an earlier study [13,16]. During the first 5 months, immune responses were comparable in both groups; at 7 months, patients vaccinated with recombinant vaccine showed a slightly higher seroconversion rate and G M T and a slightly but significantly higher proportion of vaccinees with anti-HBs concentrations above 10 IU/I (Fig. 1, Table 2). Female patients showed a slightly better immune response to recombinant vaccine than male patients: 78% of women were anti-HBs-positive at month 7, with a G M T of 224 IU/I, as compared to 62% and 142 IU/I in men. These differences, however, were not statistically significant.
RECOMBINANT HEPATITIS B VACCINE
193
TABLE 2 ANTI-HBS ONE MONTH AFTER THE THIRD VACCINATION (MONTH 7)
Mean age ( y r ) ± S D Anti-HBs-positive > 10 IU/I > 100 IU/I > 1000 IU/I > 10000 IU/I
Dialysis patients (recombinant vaccine) total(n = 49)
Healthy controls (recombinant vaccine) (n = 16)
Dialysis patientsa (plasmavaccine) (n = 75)
52.5 ± 12.9
29.3 ± 10.4
47.0±14.1
100.0% 100.0% 100.0% 62.5% 0
52.0% 42.7%* 29.4% 4.1% 0
897.4 IU/I 897.4 IU/I
10.4 IU/I 89.8 IU/I
69.4% 65.3%* 34.7% 16.3% 2.0%
Geometric mean anti-HBs all: responders:
36.8 IU/I 180.7 IU/I
" Vaccinated in an earlier study. * P = 0.014 (chi-square test).
Comparison of 6-dose and 3-dose regimens Thirty-nine patients were vaccinated 6 times at monthly intervals (months 0 - 5 ) with either 20/~g H B s A g (group II, n = 19) or 40 p g H B s A g (group III, n = 20), and the results were c o m p a r e d to those of 20 patients with similar sex and age distribution vaccinated in the same dialysis unit by a 3-dose regimen (group Ia, subgroup of group I). D u r i n g the first 6 months, all 3 groups showed a steady increase in G M T and seroconversion rates with significant differences between the 3 groups (Figs. 2 and 3) at month 6: group III showed the highest seroconver-
sion rate (72%) and G M T (69 IU/I) followed by group II (6 x 20/~g) (53% and 10.0 IU/I) and Ia (3 x 40/~g) (37% and 3.4 IU/I) ( I a v s II: P < 0.05; I a v s III: P < 0.01; II vs III: P < 0.05). 26%, 37% and 70% of vaccinees, respectively, had anti-HBs levels greater than 10 IU/1 in groups Ia, II and III at this time. H o w e v e r , the third injection in group Ia at 6 months resulted in a m a r k e d increase in seroconversion (63%) and G M T (23 IU/1) as m e a s u r e d one month later, whereas at this time the mean G M T in group II was essentially unchanged (10.8 IU/I), and had already decreased in group III (35.4 IU/I). The ANTI-HBS IU/Lt
SEROCONVERSION %, 100
1000 "1
/
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GROUP 3. 6 • 40 pg HBsAG
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~
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10~
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!
1
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4
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MONTH
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Fig. 2. Seroconversion rates of dialysis patients after vaccination with 3 x 40 lzg HBsAg (group Ia, n = 19), 6 x 20/~g
HBsAg (group 11, n = 19) or 6 x 40pg HBsAg (group ill, n = 20) of recombinant hepatitis B vaccine.
i •
•
•
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2
5
4
g
6 •
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•
•
•
"?
g
10 MONT~'I V A C C I N A T I O NGROUP S 1a
V A C C I N A T I O NGROUPS S 2 ANO 3
Fig. 3. Anti-HBs responses (GMTs, all subjects) of dialysis patients after vaccination with 3 x 40/tg HBsAg (group Ia, n = 19), 6 x 20/~g HBsAg (group II, n = 19) or 6 x 40Fg HBsAg (group III, n = 20) of recombinant hepatitis B vaccine.
194 percentages of vaccinees with anti-HBs values > 10 IU were now 55%, 42% and 74% in groups Ia, II and III. Differences in GMT, between groups Ia and III were no longer significant (P > 0.10); in fact, 3 months later group Ia showed the highest GMT. Seroconversion rates and percentages of vaccinees with anti-HBs > 10 IU were now 63% and 53% for group Ia, 53% and 53% for group II and 74% and 61% for group III.
Side-effects In general, the vaccine was tolerated very well. Besides minor local symptoms such as transient pain, burning, itching and slight swelling at the injection site or systemic symptoms including headache, nausea or dizziness, which were reported after 23 of 393 injections, no serious adverse reactions were observed. In most patients, body temperatures did not rise significantly within 5 days after each vaccination. Temperatures of 37.5 or higher were measured in 24 patients; 4 patients had fevers between 38.0 and 38.8. There was no difference in severity and frequency of symptoms in patients vaccinated 3 times or 6 times, or in patients and healthy controls.
Discussion
The aim of this study was the evaluation of a new, recombinant hepatitis B vaccine in dialysis patients. In most trials, the immune response of these patients to plasma-derived hepatitis B vaccines has been poor, which is probably due to chronic uraemia rather than to dialysis as similar results were observed in uraemic patients vaccinated before dialysis [12,13]; the cause underlying the poor response to such hepatitis B vaccines is unknown. Similarly, in our study, the seroconversion rates and G M T of 49 dialysis patients vaccinated 3 times with recombinant hepatitis B vaccine containing 40 pg HBsAg per dose were considerably lower than the values of a control group of healthy adults vaccinated with the same batch of vaccine but with 10 pg HBsAg per single dose. As there were considerable differences in mean ages between these two groups, a di-
w. JILG et al. rect comparison was not possible; however, results in our control group were similar to those obtained in healthy persons with other batches of recombinant vaccine [4] or with plasma-derived vaccine [22] demonstrating good immunogenicity of the vaccine used in the present study. The immune response of the patients was comparable to that of dialysis patients vaccinated earlier with 40 /~g/dose of plasma-derived vaccine. The proportion of patients with anti-HBs concentrations of > 10 IU/! was slightly, but significantly higher after recombinant vaccine; however, in a historical comparison, relatively small differences such as this one should not be overinterpreted. A comparison with other vaccination studies with plasma-derived vaccine carried out in patient groups of similar size and composition, shows that results obtained in our study group were indeed similar to the majority of trials, in which 53-75% of patients had anti-HBs values of more than 10 IU/I with GMTs of 85-232 IU/I [10-12,15,18]. Only two studies reported clearly higher values: the study of Benhamou et al. [17] (82% seroconversion, G M T 1123 IU/I), and the study of Desmyter et al. [20] using the semipurified and only heat-inactivated Netherlands Red Cross vaccine (90% seroconversion, G M T 1586 I.U/1). The influence of sex on the immune response was demonstrated by a slight trend to higher seroconversion rates and anti-HBs concentrations in women. However, these differences were neither statistically significant nor as pronounced as differences found in healthy subjects [21,24]. Also, as shown by other studies [11,20], the immune impairment due to chronic uraemia seems to be the overriding influence on the immune response of these patients. Since vaccination schedules using more frequent injections have been successful [13,14,17] we studied the efficiency of a multi-dose regimen in 39 patients using 6 monthly injections of either 20 or 40 pg of vaccine. However, according to our results the benefits of this schedule are doubtful because after 7 and 10 months no statistically significant differences in seroconversion and GMTs could be found in patients injected 6 times with 40 pg HBsAg compared to those vaccinated only 3 times with 40 pg; an even signifi-
195
RECOMBINANT HEPATITIS B VACCINE cantly lower G M T was seen in the 6 times 20-pg group. A similar observation was reported by Benhamou et al. [17]: a better response was noted in dialysis patients after 3 injections of a double dose than after 4 injections of the standard dose. In conclusion, although a comparison with the other studies is difficult due to the heterogeneity of age distribution, time on dialysis and underlying disease of patient groups, our results indicate that in dialysis patients r e c o m b i n a n t hepatitis B vaccine seems to be as immunogenic as the two licensed plasma-derived vaccines. Side-effects were negligible, not differing from those seen after plasma vaccine. Thus, recombinant vaccines can replace plasma-derived vaccines
References 1 Stevens CE, Taylor PE, Tong MJ, et al. Hepatitis B vaccine - - An overview. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease, Grune and Stratton, Orlando, 1984: 275-291. 2 Purcell RH, Gerin JL. Prospects for second and third generation hepatitis B vaccines. Hepatology 1985; 5: 159-163. 3 Scolnick EM, McLean AA, West DJ, et al. Clinical evaluation in healthy adults of a hepatitis B vaccine made by recombinant DNA. J Amer Med Ass 1984; 251: 2812-2815. 4 Jilg W, Lorbeer B, Schmidt M, et al. Clinical evaluation of a recombinant hepatitis B vaccine. Lancet i984; ii: 1174-1175. 5 Davidson M, Krugman S. Immunogenicity of recombinant yeast hepatitis B vaccine. Lancet 1985; i: 108-109. 6 Dandolos E, Roumeliotou-Karayannis A, Richardson SC, Papaevangelou G. Safety and immunogenicity of a recombinant hepatitis B vaccine. J Med Virol 1985; 17: 57-62. 7 Hollinger FB, Troisi CL. Anti-HBs responses to vaccination with a human hepatitis B vaccine made by recombinant DNA technology in yeast. J Infect Dis 1985; 153: 156-159. 8 Heijtink RA, Kruining J, Bakker M, Schalm SW. Immune response after vaccination with recombinant hepatitis B vaccine as compared to that after plasma-derived vaccine. Antiviral Res 1985; Suppl. I: 273-279. 9 Kuwert E, Scheiermann N, Gesemann M, et al. Dose range study in healthy volunteers of a hepatitis B vaccine produced in yeast. Antiviral Res 1985; Suppl. I: 281-288. 10 Crosnier J, Jungers P, Courouce AM, et al. Randomised placebo-controlled trial of hepatitis B surface antigen vaccine in french haemodialysis units: II, Haemodialysis patients. Lancet 1981; i: 797. 11 Goudeau A, Coursaget P, Barin F, et al. Prevention of hepatitis B by active and passive-active immunization. In: W Szmuness, HJ Alter, JE Maynard (Eds.), Viral Hepatitis. Franklin Institute Press, Philadelphia, PA, 1982: 509-525.
for dialysis patients. Nevertheless, an improvement of the i m m u n e response of these patients is needed, perhaps by increasing single doses, by the use of immuno-stimulators, by the use of more immunogenic vaccine preparations such as micelle vaccines [22] or vaccines containing the pre-S sequence of HBsAg [2].
Acknowledgements We want to thank Liane Sakreida for technical assistance and Dr. Jean B. Deinhardt for her help in preparing the manuscript.
12 Grob PJ, Binswanger U, Zaruba K, et al. Immunogenicity of a hepatitis B subunit vaccine in hemodialysis and in renal transplant recipients. Antiviral Res 1983;3: 43-52. 13 Bommer J, Deinhardt F, Jilg W, et al. Impfung ur~imischer Patienten gegen Hepatitis B, Dtsch Med Wschr 1983; 108: 1823-1826. 14 Bommer J, Grussendorf M, Jilg W, et al. Vaccination against hepatitis B in patients with renal insufficiency, Proc EDTA-ERA 1984; 21: 300-305. 15 Miiller R, Arnold W, Deinhardt F, et al. Hepatitis B Impfung von immunsupprimierten Patienten. In: F Deinhardt and H Spiess (Eds.), Impfung gegen Hepatitis B. Die Medizinische Verlagsgesellschaft, Marburg/Lahn, 1983: 51-54. 16 Jilg W, Zachoval R, Schmidt M, Deinhardt F. Aktive und passive-aktive Impfung gegen Hepatitis B. In: F Deinhardt and H Spiess (Eds.), Impfung gegen Hepatitis B. Die Medizinische Verlagsgesellschaft, Marburg/Lahn,, 1983: 39-50. 17 Benhamou E, Courouce AM, Jungen P, et al. Hepatitis B vaccine: randomised trial of immunogenicity in haemodialysis patients. Clin Nephrol 1984;21: 143-147. 18 Stevens CE, Alter HJ, Taylor PE, et al. Hepatitis B vaccine in patients receiving hemodialysis, N Engl J Med 1984; 311: 496-501. 19 World Health Organization, WHO Technical Report Series No. 638. WHO, Geneva, 1979: 29. 20 Desmyter J, Colaert J, Comparative immunogenicity of MSD, Pasteur and CLB hepatitis B vaccines in 245 hemodialysis patients. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stranon, Orlando, 1984: 709-710. 21 Zachoval R, Jilg W, Lorbeer B, et al. Passive/active immunization against hepatitis B. J Infect Dis 1984; 150: 112-117. 22 Howard CR, Skelly J, Tsiquaye KN, et al. The development and properties of alternative hepatitis B polypeptide vaccines. In: GN Vyas, JL Dienstag and JH Hoofnagle (Eds.), Viral Hepatitis and Liver Disease. Grune and Stratton, Orlando, 1984: 410-423.