Simultaneous passive and active immunization against hepatitis A A. L e e n t v a a r - K u i j p e r s *+, R. A . C o u t i n h o * ,
V. B r u l e i n ++a n d A. S a f a r y ++
The serum antibody response to simultaneous administration of immune globulin ( Ig ) and an inactivated hepatitis A vaccine was investigated in health), volunteers who had been tested and found free of hepatitis A virus. One hundred and jorty nine subjects were randomly allocated into three groups. Group 1 received three doses of hepatitis A vaccine at O, 1 and6 months, group 2 received5 ml of lg and group 3 received a combination of lg and vaccine. In group 3 the seropositivity rate measured by enzyme-linked immunosorbent assay was 100% at day 5, month 1 and 2, 96% at month 6 and again 100% at month 7. In the group that received vaccine alone the seroconversion rates were O, 96, 100, 98 and 100 respectively. The geometric mean titres in subjects who received passive/active immunization were about twofold lower than in subjects who received vaccine alone, indicating interference of Ig with the immune response. Despite this, the data show that simultaneous administration of hepatitis A vaccine and Ig confers both immediate protection via Ig administration and long-term vaccine-induced protection. As the antibody levels reached are about twofold lower compared to that after administration of hepatitis A vaccine alone, a booster dose may be required sooner, than if the vaccine were administered alone. Keywords: HAV vaccine; immune globulin; immunogenicity; interference
INTRODUCTION The efficacy of immune globulin Ig in preventing hepatitis A has been frequently documented. Administration of Ig provides rapid protection that is limited, however, to five months after injection ~-S. Following successful cell culture propagation of hepatitis A virus (HAV) by Provost et al., several candidate vaccines have been developed inducing antibodies that persist for a long period of time ~ m. The antibody response and persistence of antibodies have been shown to be related to the potency of the vaccine dose and to the number of doses administered ~t ~3. High doses induced earlier and more frequent seroconversion. However, a primary response can only be expected at the earliest 8 to 10 days after administration of the vaccine. In circumstances where susceptible individuals may rapidly be placed at risk of hepatitis A through, for example travel to endemic areas or contact with a hepatitis A patient, immediate protection is warranted. In these situations simultaneous administration of passive and active immunoprophylaxis would be indicated provided that Ig does not hamper the immune response to the vaccine. The present study was therefore undertaken to evaluate the reactogenicity and the immunogenicity of an
*Municipal Health Service, PO Box 20244, 1000 HE Amsterdam, The Netherlands. *SmithKline Beecham Biologicals, Rixensart, Belgium. tTo whom correspondence should be addressed
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inactivated hepatitis A vaccine in healthy adults, alone and when given simultaneously with Ig. MATERIALS AND METHODS
Vaccine / immune serum globulin The vaccine was prepared from the HMI75 strain (RIT 4380) grown on MRC-5 cells. The virus was purified and inactivated as described previously ~4. Each dose was calculated to contain 720 ELISA units of hepatitis A antigen (measured by enzyme-linked immunosorbent assay, ELISA) adsorbed on to 0.5 mg aluminium hydroxide in a volume of 1 ml. The immune serum globulin (Ig) used was obtained from the Dutch Red Cross. It contained 10(>200 IU/ml specific anti-HAV immunoglobulin. Serology Serum liver enzyme activities were measured using an automated kinetic method. Antibodies against HAV were measured using an ELISA inhibition assay, developed by SmithKline Beecham Biologicals. Microwell plates are coated overnight at room temperature with a purified human IgG anti-HAV. Alter saturation of the wells with 1% gelatine, purified inactivated HAV is added and the plates incubated for 1 hour at 37°C. Serum from vaccinees is then added in twofold dilutions and the plates incubated overnight at room temperature. Human lgG anti-HAV, identical to that used to coat the plates was conjugated with peroxidase. 0264-410X/92/100S138-04 © 1992Butterworth-HeinemannLid
Passive~active immunization against hepatitis A: A. Leentvaar-Kuijpers
After addition of the conjugate, the plates are further incubated for 1 hour at 37°C. The absorbance at 490 nm of each well is read following addition of the peroxidase substrate (o-phenylene diamine dihydrochloride in citrate buffer containing H20:). Anti-HAV titres are calculated in mIU/ml, in comparison with an immunoglobulin preparation (WHO standard, calibrated at 100 IU) using the four parameter method ~5. Sera with antibody titres > 20 mlU/ml are considered positive. Samples showing aspecific interference was titrated using a commercially available radioimmunoassay (RIA; HAVAB, Abbott Laboratories, Chicago). Samples which inhibited the binding by > 20% were considered positive.
in group 2 only), and 6. Subjects in group 1 and 3 also had blood taken at 7 and 12 months.
Reporting of signs and symptoms after injection At 3 and 8 h after injection and once a day for the three subsequent days, subjects were asked to record general signs and symptoms and local signs and symptoms on a symptom sheet provided.
Analysis of results The Z 2 test and Students' t test, as indicated, were used to compare the results in each group. RESULTS
Study participants
Characteristics of the study population
After approval of the study protocol by the Ethics Committee of the Amsterdam, Municipal Health Service volunteers between the ages of 18 and 50 years were recruited, screened and selected. They had to give written informed consent, to be in good general health, to have normal levels of serum aspartate aminotransferase and alinine amino-transferase and to be negative for antiHAV antibodies on serum samples taken 14 days before receiving vaccine or Ig.
The 149 volunteers, comprised of 62 males and 87 females, were between the ages of 18 and 49 years (mean age 30.3 years; S.D. = 7.98 years). There were no significant differences in the mean ages or the sex ratio in the three groups.
Immunization and blood sampling One hundred and forty-nine subjects were randomly allocated into three groups to receive either vaccine alone, Ig alone or both vaccine and Ig, according to the scheme outlined in Table 1. The vaccine was administered in the deltoid region intramuscularly. A volume of 5 ml Ig was injected intramuscularly in the upper thigh. Blood samples were taken from all subjects on day 5 and months 1,2, 5 (for subjects Table 1
Scheme for administration of vaccine, Ig and the combination
Group
No. of subjects
Prophylaxis
1
50
2 3
49 50
vaccine at 0, 1 months + booster vaccine at 6 months Ig at month 0 vaccine at 0, 1 months + booster vaccine at 6 months + Ig at month 0
Table 2
Safety and reactogenicity Serum liver enzyme levels One subject in group I, five subjects in group 2 and three subjects in group 3 had elevated alanine aminotransferase (ALT) levels (>_ 45 IU/I) at various time points during the study period. These increased levels occurred sporadically. No clinically significant long-term increases were observed.
Solicited signs and symptoms In total, 98% of the symptom sheets were returned. No serious adverse reactions were reported by any subject. Minor signs and symptoms were reported by 76% of the subjects in group 1, 84% in group 2 and 92% in group 3 after the first injection (Table 2). The higher percentage of subjects with symptoms in group 3 compared to group 1 was due to an increase in the reporting of local symptoms. The frequency of adverse reactions after the second and booster dose of vaccine in group i and 3 was lower compared to that after the first vaccine dose. The most freqently reported local reaction after hepatitis A vaccine administration was soreness (49%, 146/297 doses). Approximately one-half of all these cases lasted no longer that
Subjects with/without symptoms and the nature of symptoms reported Symptoms
Dose
1
2 3 Overall
Group No.
1 2 3 1 3 1 3 1 2 3
Local After HAV
Local After Ig a
General
Any
N
n
%
n
%
n
%
N
%
50 49 50 50 50 49 48 149 49 148
34 28 26 20 24 27 84 75
68 56 52 40 49 56 56 51
39 42 39 42
80 84 80 84
14 12 13 8 8 7 13 29 12 34
28 25 26 16 16 14 27 20 25 23
38 41 46 30 23 27 32 95 41 101
76 84 92 60 46 55 67 64 64 68
N, Total number of sheets returned following a given vaccine dose and overall; n, number of sheets with or without symptoms reported following a given vaccine dose and overall. Results for local symptoms is the percentage of subjects reporting at least one local symptom in area injected with HAV or Ig. Results for general symptoms are the percentage of subjects reporting at least one general symptom
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Passive/active immunization against hepatitis A." A. Leentvaar-Kuijpers
24 h. Other local reactions were reported with a frequency of 18% or less. After injection of Ig soreness was also the most frequently reported symptom (75%, 74/99 injections). Headache was the most frequent reported general symptom. The frequency of occurrence after the first vaccine dose was 18% and 12% in group 1 and 3, respectively. After the second dose the percentage was 10 in both groups. This was identical to that observed after the administration of Ig. After the third dose the frequency was 6% in group 1 and 10% in group 3. Three subjects reported 'severe' general symptoms i.e severe vomiting following the first vaccine dose in group 1, severe headache and vomiting after administration of Ig and severe headache after the booster dose of vaccine in group 3.
Comparison of the antibody levels after administration of vaccine and/or Ig All subjects in group 2 and 3 had anti-HAV antibodies in the first postinjection blood sample at day 5. No subject in group 1 receiving vaccine alone had seroconverted (Table 3). At day 5 the geometric mean titres Table 3 Seropositivity and geometric mean anti-HAY antibody titres (GMT) of seropositive subjects Postvaccination injection Group No.
Sample at a
N
Anti-HAV+
Seropositive (%)
1
5d 1 me 2me 6 me 7 me 5d 1 me 2 me 5 me 6 me 5 d 1 me 2me 6 me 7 me
45 49 49 48 45 49 49 48 47 47 48 47 47 46 43
0 47 49 47 45 49 49 42 5 0 48 47 47 44 42
0 96 100 98 100 100 100 88 11 0 100 100 100 96 98
2
3
I II II III IV I I II III IV 1 I II III IV
GMT
236 563 364 3967 145 78 66 42 155 136 209 165 2211
aTime of blood sample from vaccination Table 4
(GMTs) in groups 2 and 3 were, respectively 146 and 155 mlU,'ml One month after the primary vaccination course, all subjects that had received vaccine (groups 1 and 3) were anti-HAV antibody positive. However, the G M T for group 1 was significantly higher than that of group 3(563 mlUi'ml compared to 209 mlU/ml). The G M T s of groups 1 and 3 were significantly higher than those of subjects in group 2, who had received only lg. During the follow-up period, the subjects in group 2 continued to lose antibody. At month 5. only five of the 47 subjects who returned for blood sampling were antiHAV antibody-positive: by month 6, no subject in this group had detectable titres of antibody. In the two groups which had received vaccine, only three subjects (one in group I and two in group 3) were seronegative at month 6. The booster dose of vaccine elicited antibodies in all of these subjects. One subject in group 3 did not respond to the booster and was seronegative for antiHAV antibodies at month 7, even though he had responded to the first and second dose of vaccine. The booster increased the G M T in group 1 from 364 to 3967 mIU/ml at month 7, a > 10-fold increase. For group 3, a 13.4-fold increase was observed, with G M T s of 165 and 221 I m l U / m l at months 6 and 7, respectively. As was demonstrated for the first and second vaccine dose, the G M T of group 1 was significantly higher than that of group 3 one month after the booster dose (p = 0.005 by Students' t test). The distribution of individual antibody titres is given in Table 4. One month after the primary vaccination course, 88% (43/'49) of the subjects in group I had titres > 200 mlU/ml, which is approximately equal to the G M T obtained five days after the administration of lg. In group 3, 53% (25/47) of the subjects were above this level. At month 7, all seropositive subjects in both groups 1 and 3 were above this level. Three subjects (one from group 1, two from group 3) who were not included in this analysis, due to irregular vaccination or blood sampling schedules, had anti-HAV antibody titres > 2000 mlU,,'ml at month 7. These subjects, who were for the same reasons excluded from the follow-up, at months 6 and 7 only had titres _> 200 m l U /
Distribution of individual anti-HAY antibody titres (mlU/ml) Postvaccination injection
Titre _200 < 2000
_>2000
Group
No.
Sample at a
N
n
%
n
%
n
%
n
%
1
I I II III IV I I II III IV I I II III IV
5{:1 1m o 2me 6me 7me 5d 1 me 2me 5me 6me 5d 1 me 2me 6me 7me
45 49 49 48 45 49 49 48 47 47 48 47 47 46 43
45 2 0 1 0 0 0 6 42 47 0 0 0 2 1
100 4 0 2 0 0 0 13 89 100 0 0 0 4 2
0 18 6 10 0 40 48 42 5 0 33 36 22 28 0
0 37 13 21 0 82 98 88 11 0 69 77 47 61 0
0 29 40 36 11 9 1 0 0 0 15 11 25 16 15
0 59 81 75 24 18 2 0 0 0 31 23 53 35 35
0 0 3 1 34 0 0 0 0 0 0 0 0 0 27
0 0 6 2 76 0 0 0 0 0 0 0 0 0 63
2
3
,Time of blood sample from vaccination. N, Total number-sampled; n, number of samples with defined titre
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Passive/active i m m u n i z a t i o n against hepatitis A: A. Leentvaar-Kuijpers
ml at month 7. Exclusion of these subjects did not affect the age/sex distribution in the three groups. DISCUSSION AND CONCLUSION The vaccine used in this study was shown to be safe and well tolerated. Following vaccination no serious adverse reactions were reported. Reactions reported were mild. There were no clinically significant changes in liver enzyme activities. Moreover, the frequency of elevation in serum liver enzyme activities was lower in recipients of the vaccine compared to that in Ig recipients. The elevations were mild and occurred sporadically. The lower frequency of reporting of reactions after the second and third vaccine dose compared to the first indicates that hypsersensitivty was not induced. Simultaneous administration of vaccine and Ig did not result in an increase in systemic reactions in comparison with either the vaccine or Ig alone. The vaccine was also shown to be highly immunogenic. All of the seronegative volunteers had seroconverted one month after the second dose. The vaccine induced higher G M T s than did Ig administration. This was particularly encouraging since a high dose of Ig was used in the study (i.e. a dose shown to provide passive protection against hepatitis A for 4-6 months). Although all subjects who received vaccine and Ig simultaneously (group 3) responded to the vaccine, they had lower anti-HAV antibody titres on average than did subjects administered vaccine alone, indicating interference of the Ig with the immune response. The booster dose of vaccine resulted in a > 10-fold increase in anti-HAV antibody titre, as measured one month after this dose in almost all subjects. However, since antibody levels reached are about twofold lower in subjects receiving passive-active immunization compared to subjects receiving vaccine alone, protective antibody levels may persist for a shorter period of time compared to that after administration of vaccine alone. Following passive-active immunization a booster dose may be required sooner. In conclusion, the inactivated hepatitis A vaccine used in this study was shown to be well-tolerated and to induce high titres of anti-HAV antibody. The strong
immune response elicited by the vaccine, even when simultaneously injected with high doses of Ig, indicates that passive/active immunication of persons who are or soon will be exposed to HAV is readily achievable.
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