Original Articles Time Course of Hepatitis A Virus Antibody Titer after Active and Passive Immunization SHIGETOSHI FUJIYAMA,' SHIRO IINO,' KOICHIODOH,3 SHOJI KUZUHARA,3HIROAKIWATANABE,3 MAsAHIKo TANAKA,~ KYOSUKE MIZUN03 AND TATSUO SATO' IThird Department of Internal Medicine, Kumamoto University Medical School, Kumamoto 860; 2First Department of Internal Medicine, Faculty of Medicine, Tokyo University, Tokyo 113; and 3The Chemo-Sero-Therapeutic Research Institute, Kumanoto 869-12, Japan
To investigate the antibody titer necessary to inactivated adjuvant-free vaccine using the cell line prevent hepatitis A virus infection, either 15 or 7.5 GL37 and the HAV strain KRM003 (4). The vaccine mg/kg of immune serum globulin was injected into 10 consists of a suspension of 1 p,g/ml purified virus antihepatitis A virus negative volunteers and their (confirmed by ELISA) that is supplemented with preserum antihepatitis A virus titers were observed for 28 servatives and a vehicle before being freeze-dried. Phase wk. In addition, antibody titers were observed for 96 1 clinical trials started in July 1988 and have already wk in a phase 1 clinical trial of a hepatitis A vaccine. concluded. This vaccine has shown a good immunogeThe two studies were then compared to assess the immunogenicity of the vaccine and the persistence of nicity and produces a dose-dependent antibody response the antibody. Serum-neutralizing antibody titers that without side effects in humans (5). However, it was were greater than or equal to 4 (consideredas positive) difficult to conduct efficacy studies to determine the persisted for 18 wk and 14 wk after the injection of 15 minimum effective antibody titer necessary to prevent and 7.5 mg/kg of globulin, respectively. Hepatitis A HAV infection because no endemic areas for hepatitis A virus vaccine recipients showed adequate neutralizing were left in Japan. antibody titers, with the groups receiving 1, 0.5 and In this study, intramuscular ISG was given to 10 0.25 pg/dose showing titers of 46*5,44.7and 44, respec- anti-HAV-negative healthy male volunteers to predict tively, at 18 mo after the third inoculation. These the antibody titer necessary to preventing HAV infindings suggested that effective blood antibody titers were likely to be retained in the 1.0 kg or 0.5 Fg/dose fection. Their serum anti-HAV titers were observed for groups for at least several years. Moreover, the serum 28 wk. We also observed anti-HAV titers during the antihepatitis A virus titers demonstratedby a modified long-term follow-up of a group of hepatitis A vaccine radioimmunoassay changed in parallel with the neu- recipients. By comparing these two groups, the duration tralizing antibodytiters in the volunteers injectedwith of the effective antibody response and the immunogeglobulin. (HEPATOLOGY 1992;15983-988.) nicity of the vaccine were estimated.
Human immune serum globulin (ISG) has long been used prophylactically to prevent hepatitis A virus (HAV) infection, mainly for those people traveling abroad to areas where the virus is endemic or for close contacts of known hepatitis A cases (1).To improve the efficacy of ISG against hepatitis A, standard ISG preparations have been developed by the World Health Organization (WHO) (2). However, because the effective duration of the passive immunity afforded by ISG is limited by its serum half-life of 21 days or less (3) and because it is feared that the antibody concentration in ISG preparations may be decreasing, renewed calls have been heard for the development of a hepatitis A vaccine. In Japan, work has been undertaken to develop an Received July 10, 1991; accepted January 10, 1992. Address reprint requests to: Shigetoshi Fujiyama, M.D., Third Department of Internal Medicine, Kumamoto University Medical School, 1-1,Honjo 1-chome, Kumamoto 860, Japan. 31/1/36488
MATERIALS AND METHODS ?Globulin Trial. ?-Globulin (Lot No. 129-B) prepared by The Chemo-Sero-TherapeuticResearch Institute (Kumamoto, Japan) and containing 150 mg/ml of immune globulin was injected into the gluteus muscle in two groups of five male volunteers at a dose of 15 mgkg (group A) or 7.5 mgkg (group B). All the volunteers (mean age = 26.9 ? 3.0 yr, range = 23 to 34 yr) were negative for antibody to HAV. Blood samples were taken 1, 2, 3, 4, 5 and 7 days and 2, 3, 4, 6, 8, 10, 13, 16, 20, 24 and 28 wk after injection. Long-term Follow-up of Antibody Titers after Hepatitis A Vaccination. In the phase 1 clinical trial, hepatitis A vaccine
(Lot No. K-02, prepared by the above-mentionedinstitute) was injected intramuscularly into the upper arm in three groups of four healthy volunteers (mean age = 31.3 k 4.6 yr, range = 26 to 41 yr) who were negative for anti-HAV.The dose given was 0.25 ml (0.25 pg), 0.5 ml (0.5 pg) or 1.0 ml (1.0 pg), and the injection was performed at time zero and 1 and 6 mo later. The acquired anti-HAV titer was then measured at frequent intervals until the thirty-second week after the first injection. Subsequently, blood samples were collected at 48,72 and 96 wk to assess long-term antibody retention.
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FUJIYAMA ET AL.
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TABLE1. HAV antibody titers of human immune serum globulin Manufacturer
Lot No.
ELISA (mIUim1)"
N.T.b
HAVAB RIA"
Ad
918 919 920
121,000 129,000 120,000
1: 174,000 1:256,000 1: 195,000
1:800 1:663 1: 726
B'
050 052
30,000 33,000
1:64,000 1:48,000
1:154 1:217
Cf
126 127 129 131
155,000 118,000 126,000 139,000
1:182,000 1: 151,000 1: 169,000 1: 178,000
1: 721 1:800 1:668 1:711
WHO reference
No. F
100,000
1:600 (1: 442-1 : 762)
"Competitive inhibition ELISA. The antibody titer was calculated from the ratio of the 50% competitive inhibition titer for the simultaneously reacted positive control (2,000 mIU/ml) t o that of the test sample. bNeutralizing antibody titer (N.T.) was measured by ELISA using a GL37 monolayer cell sheet in a 96-well plate. 'Antibody titer was measured according to the procedure outlined for the HAVAB RIA kit (Abbott Laboratories, North Chicago, IL). dNihon Pharmaceutical Co. Ltd. (Tokyo, Japan). 'Japanese Red Cross (Tokyo, Japan). fThe Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). gDev Biol Stand 1983;54:411-416.
Measurement of Anti-HAV Antibody Titers. Serum anti-HAV antibody titers were assessed by three of the following four methods. First was the competitive inhibition ELISA. The principle of this ELISA was similar to that of the HAVAB-EIA test (Abbott Laboratories, North Chicago, IL). Test samples were diluted with PBS (pH 7.2) containing 0.2% BSA and 0.05% Tween 20. Fourfold serial-diluted test samples and enzyme-labeled antibodies were reacted in antigen-coated plates. After washing, the enzyme substrate was added and color was developed. The antibody titer was calculated from the ratio of the 50% competitive inhibition titer for the simultaneously reacted positive control (2,000 mIU/ml, adjusted at the National Institutes of Health using WHO reference preparations) and that of the test sample. The second method was the neutralizing antibody assay. Neutralizing antibodies were measured by an ELISA using an HAV-sensitive GL37 monolayer cell sheet grown on a 96-well plate. Test samples were diluted with PBS (pH 7.2) containing 2% FBS and 0.002%Tween 80 (gentamicin was added at a final concentration of 50 pg/ml). Fourfold serial-diluted test samples and HAV at 50 to 100 median tissue culture infective doses (TCID,,)/ml were incubated at 37" C for 3 hr and then overnight at 4" C, after which each sample was added to four wells of the 96-well plate containing the GL37 monolayer and cultured. Two weeks later the cells were washed with PBS (pH 7.2) and fixed in 80% methanol supplemented with 30% hydrogen peroxide (1: 1,000). Then 50 pl of anti-HAV rabbit serum was added to each well, and the plates were incubated for 1 hr at 37" C. After further washing, 5Opl of horseradishconjugated goat antirabbit IgG (Medical and Biological Labs Co., Ltd., Nagoya, Japan) was added to each well, and the plates were incubated for 2 hr at 37" C. After final washing, the enzyme substrate was added and color was developed. Inhibition of more than 70% of the absorbance of the virus control was determined to be positive. The dilution that exhibited 50% neutralization was calculated by the method of Reed and Muench (6) and was defined as the neutralizing titer. The cutoff level was determined to be 4. The third method was anti-HAV RIA. Antibody titers were
measured according to the procedures outlined in the instructions of the HAVAJ3 RIA kit (Abbott Laboratories). This standard RIA was used to measure anti-HAV levels in the volunteers injected with vaccine. Commercial human ISG preparations were serially diluted from 1: 50 with PBS (pH 7.21, and the 50% competitive inhibition titer was determined. The fourth method was modified anti-HAV RIA. We also modified the RIA so that 0.1 ml of the test sample was used to compete with 0.1 ml of radiolabeled antibody observing the method of Provost et al. (7). This modified assay was used to measure anti-HAV levels in the volunteers injected with ISG. Informed consent was obtained from each volunteer before enrollment. This study was approved by the Ethics Committee of The Chemo-Sero-Therapeutic Research Institute.
RESULTS Anti-HAV Titers in Commercial Human ISG Preparations. Table 1summarizes the anti-HAV titers of the
globulin preparations commercially available in Japan, as determined by ELISA, neutralization and RIA. The WHO reference values obtained from the literature are also included. The globulin preparations studied consisted of a total of nine lots produced by three companies. Imported plasma was used by companies A and C , and domestic plasma obtained from blood donors was used by company B. The two lots from company B showed antibody titers that were approximately one fourth of those in the lots from companies A and C , regardless of the method of measurement used. The lots provided by companies A and C had titers exceeding those of the WHO reference preparations, whereas the lots from company B showed lower titers. Lot-129 from company C (used in this intramuscular globulin trial) gave an ELISA level of 126 IU/ml, a neutralizing titer of 1:169,000 and an RIA titer of 1:668. Comparison of the three measurement methods
Vol. 15, No. 6, 1992
ANTI-HAV TITER AFTER ACTIVE OR PASSIVE IMMUNIZATION
showed that an anti-HAV titer of 1 IUiml corresponded to a neutralizing titer of 1:1,500 and a n RIA titer of 1:6. Time Course Changes of the Neutralizing Antibody Titer after Injection ofISG or Vaccination. The changes
in neutralizing antibody titers in the three vaccination groups and the two groups injected with ISG are shown in Figure 1. The half-life of the neutralizing antibody after ISG injection was 20 days in group A and 19 days in group B. Neutralizing titers above 4 (considered as positive) were respectively noted until 18and 14 wk after injection in groups A and B. Effective neutralizing antibody titers were achieved in the subjects inoculated with vaccine. In the groups receiving 1.0, 0.5 and 0.25 pgidose, the neutralizing antibody titers were respectively 43.9,44.0and 4'.O at 8 wk (1mo after the second inoculation); 43.7,43.2and 41.7 at 24 wk (5 mo after the second inoculation); 46.9, 45.9 and 45.4 at 28 wk (1 mo after the third inoculation); and 45.5,44.7and 44 at 96 wk (18 mo after the third inoculation). We compared the antibody neutralizing activity of serum obtained from vaccine recipients with that of the ISG preparations. Serum samples from vaccine recipients and samples of the ISG preparations were diluted and adjusted to ELISA values of 100, 10, 1 and 0.1 mIU/ml and then reacted with equal volumes of 102.6, 103.6 and 104.6 TCID,,/ml HAV suspensions. This mixture was then added to 8-well cell sheets, and the neutralizing activity was expressed as a percentage of the neutralizing-positive wells (Fig. 2). Serum samples were obtained 1mo after the third injection (28 wk) from subject No. 1of the 1.0 kg/dose group and subject No. 8 of the 0.5 pgidose group. All the serum samples from the two vaccine recipients and the two lots of ISG tested showed a similar HAV-neutralizing activity.
985
ISG
0 v1 v2
0
v3
24
48
72
96
weeks after the first injection
FIG. 1. Time course of neutralizing antibody titers in the three vaccinated groups ( 0 = 1.0 yg, = 0.5 yg, = 0.25 kg) and the two groups injected with ISG ( 0 = 15 mgkg, A = 7.5 mgkg).
and thus clinical efficacy trials and the determination of the minimum effective antibody titer are both difficult to perform. ISG has been used to prevent hepatitis A since 1945, and many reports have described its effectiveness (8-10). Direct proof of the efficacy of ISG was obtained in a human inoculation trial conducted by Krugman (11)in institutionalized children with mental retardation. However, among the various reports detailing the effectiveness of ISG in the prevention of hepatitis A, only a few have described the anti-HAV antibody titers of the ISG lots used. In a study of Japanese residents in Time Course of anti-HAV Titers after Iqjection of ISG Algeria, Arakawa et al. (12) used ISG lot Nos. 878, 883 or Vaccination a s Determined by RIA. The changes in and 884 from company A that had HAVAB RIA titers of the mean anti-HAV levels determined by RIA in the 1:400 to 1:800. The ISG preparation used in this study three vaccinated groups are shown in Figure 3. All the had a titer of 1:668 by RIA. Accordingly, the difference volunteers seroconverted to anti-HAV ( 2 50% inhi- in titers is not considered sufficient to influence the bition) at 1mo after the third vaccination, and an overall effectiveness of the preparations. Moreover, the titer of dose-dependence of the response was confirmed. The the ISG used exceeded that of the WHO reference changes in the mean anti-HAV levels determined by the preparations, which are adjusted t o ensure their effecmodified RIA are shown in Figure 4 for the two groups tiveness against HAV. The blood-neutralizing antibody injected with ISG. The mean inhibition value f 2 50%; titers of the subjects given ISG showed a half-life of 20 considered as positive) was retained for 11 and 8 wk, and 19 days, respectively, in the 15 and 7.5 mgkg respectively, in the 15 mgkg and 7.5 mgkg groups. groups, and these values are in close agreement with Titration of the anti-HAV titers in two different lots of those reported in the literature (3).If a neutralizing titer ISG using the standard and the modified RIAprocedures above 1:4 is considered positive, the duration of effective is shown in Figure 5 . The sensitivity of the modified antibody retention in the 15 and 7.5 mgkg groups was assay was about 10-fold greater than that of the 18 and 14 wk, respectively. These periods are similar to standard assay, and the 50% inhibition level in the the reported 2-mo duration of protection achieved with modified assay corresponded to an ELISA value of about a dose of 0.026 ml/kg (121, the 4-mo period achieved with 15 mIU/ml. a dose of 0.05 to 0.09 mVkg (13) and the 3-mo period achieved with the dose of 0.05 m l k g stipulated in the DISCUSSION Japanese guidelines for prophylaxis against hepatitis A Important issues to be considered in the development (14). of an inactivated hepatitis A vaccine are the appropriate Although many other studies of an even longer methods and criteria for evaluating its efficacy in duration of protection have been conducted (15, 16), humans. In Japan, no areas have been seen where those reports are difficult to evaluate because the large-scale outbreaks of hepatitis A are likely to occur, antibody titers of the ISG used were not stated.
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FUJlYAMA ET AL.
986
ISG 127 100
10"TCID.Jml 10 '' TCIDr)rnl l o A 6TCIDdrnl
80 60 40
20
c
-
n
01
1
10
100
10
100
ISG 131
.-
e = 6 C
0.1
1
Vaccine (No.1) T28 100
80 c
* 0
60
m
40
c 0 0 -
20
0) c
n
a, a
01
1
10
100
Vaccine ( No.8 ) T28 100
80
60 40
20 0 01
1
10
100
HAV antibody titer (rnlUlrnl)
FIG.2. Neutralizing activity of the antibodies elicited by ISG and the vaccine. Two different lots of ISG (ISG 127 and ISG 131)were produced by company C. Two serum samples (No. 1 T28 and No. 8 T28) were obtained 1 mo after the third injection (28 wk)from subject No. 1 of the 1.0 pgldose group and subject No. 8 of the 0.5 pgldose group. A neutralization test to compare No. 8 T28 and HAV 102-6TCID,,/ml was not conducted.
Accordingly, in this study a neutralizing titer of 1:4 was taken to represent the antibody titer providing effective prophylaxis against hepatitis A, with a neutralizing titer of 1:4 corresponding to an ELISA result of 2 to 3 mIU/ml. Furthermore, in uztro cell culture tests showed that antibodies qualitatively equivalent to those found in ISG were produced when the vaccine was administered three times. Our findings indicated that the administration of the vaccine at 0.5 pg/dose or more would produce a high antibody titer for at least 5 mo after the second injection and for at least several years after the third injection (11, 17). Thus this study demonstrated that a third injection was probably effective in producing the long-term persistence of a protective antibody titer. One previous report, by Stapleton et al. (18),described the titers of neutralizing antibody to HAV in ISG preparations or in the serum of human ISG recipients. Because t,heir method of measurement differed from
ours, direct comparison of the two studies is difficult, but their ISG apparently possessed a lower antibody titer than that of the WHO reference preparations. They reported that when 18 healthy men had ISG administered at 0.02 mlkg all the serum samples collected on the third day and fifty fifth-day after injection contained detectable levels of neutralizing antibody. Both the report of Stapleton et al. (18) and our own results indicate that the neutralizing antibody titer provides good evidence of the value of ISG in the prophylaxis of hepatitis A and suggest that this test should also be used in evaluating the efficacy of hepatitis A vaccines. However, it is not a quick diagnostic test suitable for routine clinical use. We measured serum anti-HAV titers after vaccination by using the standard HAVAB RIA and assessed the titers after ISG injection by using a modified RIA. This modified assay appeared to permit about a 10-fold greater sensitivity of anti-HAV detection, a result that is
Vol. 15, No. 6, 1992 V l v2
v3
44
4
100 ,
0
'
0
'
'
'
987
ANTI-HAV TITER AFTER ACTIVE OR PASSIVE IMMUNIZATION
'
,
ISG
'
24
4,
100
,
,
,
,
,
48
.
'
.
'
,
T
'
72
,
'
,
4
0
,
12
8
96
20
16
24
28
weeks after the first injection
weeks after the first injection
FIG.3. Time course of HAV antibody titers in the three vaccination groups as determined by HAVAB RIA (0 = 1.0 pg, A = 0.5 p,g, 0 = 0.25 pg).
in agreement with the report of Provost et al. (7). In the present study, the 50% inhibition level in this modified RIA corresponded to an ELISA value of about 15 mIU/ml, indicating a n anti-HAV titer considerably higher than the 2 to 3 mIU/ml hypothesized to be necessary to prevent HAV infection. As compared with the standard assay, the modified assay had problems in terms of specificity. However, if the modified assay could be improved, its simplicity would allow its wider application in further vaccine studies. Measurement of the anti-HAV antibody titers in the ISG preparations sold by three different manufacturers demonstrated large differences related to the source of the plasma used. The antibody titers of the ISG made from imported plasma by two manufacturers were approximately the same and exceeded the anti-HAV antibody titers of the WHO reference preparations, whereas the titer of the ISG made from domestic blood donor plasma was lower than the WHO reference level and only one fourth of the titer in the other commercial ISG preparations. These results were not unexpected because the proportion of the population younger than 40 yr of age possessing anti-HAV is extremely low in Japan, and thus y-globulin preparations derived from Japanese blood donor plasma are likely to have low titers. Because Japan is tending to increasingly rely on domestic blood donor plasma as the sole source of all blood products, it is believed that a vaccine for the prevention of hepatitis A will become even more necessary in the future. The results of this study suggest that the recommended ISG dose for the prevention of hepatitis A should be decided on the basis of the anti-HAV antibody titer of each ISG lot and our data on the duration of effective antibody retention.
Acknowledgments: We wish to thank Ms. Mikiko Saito and Ms. Keiko Morozumi for their excellent technical assistance.
FIG.4. Time course of HAV antibody titers in the two groups injected with ISG as determined by the modified HAVAB RIA (0 = 15 mgikg, A = 7.5 mgkg).
100
1
t 0
h
e
6
I
A Q 0
0
I1 1
0
I
A 0
,
,
,
,..,., 10
,
.
,,....,
,
100
,
,
,...., 1000
,
,
, , , ,
J
10000
HAV antibody titer (mlUlml)
FIG.5. Titration of the hepatitis A antibody titer in two different lots of ISG (Lot 129 and Lot 131, which are represented respectively by triangles and circles) using the standard HAVAB RIA (open) and the modified HAVAB RIA (closed).
REFERENCES 1. Pollock TM, Reid D. Immunoglobulin for the prevention of infectious hepatitis in persons working overseas. Lancet 1969;1: 281-283. 2. Gerety RJ,SmallwoodLA, Finlayson JS, Tabor E. Standardization of the antibody to hepatitis A virus (anti-HAV) content of immunoglobulin. Dev Biol Stand 1983;54:411-416. 3. Wells JV.Metabolism of immunoglobulins. In: Fudenberg HH, Stites DP, Caldwell JL, Wells JV,eds. Basic and clinical immunology. Tokyo: Maruzen Co., 1976:195-203. 4. Moritsugu Y, Totsuka A. Hepatitis A vaccine: current status of development. In: Fukai K, ed. Virus vaccines in Asian countries. Tokyo: University of Tokyo Press, 1986:193-201. 5. Iino S, Fujiyama S, Horiuchi K, J o K. Clinical trial of inactivated hepatitis A vaccine [in Japanese]. Jpn J Gastroenterol 1990;87: 1532-1536. Muench H. A simple method of estimating fifty per cent 6. Reed U, endpoints. Am J Hyg 1938;27:493-497. 7. Provost PJ, Hughes JV,Miller WJ, Giesa PA, Banker FS, Emini EA. An inactivated heuatitis A viral vaccine of cell culture oripin. J Med Virol 1986;19:i3-31.
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8. Conrad ME, Lemon SM. Prevention of endemic icteric viral hepatitis by administration of immune serum gamma globulin. J Infect Dis 1987;156:56-63. 9. Green MS, Dotan K. Epidemiology: efficacy of immune serum globulin in an outbreak of hepatitis A virus infection in adults. J Infect 1988;17:265-270. 10. Green MS, Block C. Apparent effect of immune serum globulin prophylaxis in the military on viral hepatitis incidence in the civilian population in Israel. J Epidemiol Community Health 1989;43:187-190. 11. Krugman S. Effect of human immune serum globulin on infectivity of hepatitis A virus. J Infect Dis 1976;134:70-74. 12. Arakawa Y, Katsuhara N, Amaki S, Kaneda H, Honda T, Kanda Y, Satoh Y , et al. Assessment of human immune serum globulin in preventing hepatitis A infection among Japanese residents abroad [in Japanese]. Acta Hepatol Jap 1981;22:943-953. 13. Ohara H, Ebisawa I, Ohtani S. Prophylactic efficacy of immune serum globulin against hepatitis A. Jpn J Exp Med 1986;56: 229-233.
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14. Research Group of Hepatitis A. Countermeasure of prophylaxis. In: Ichida F, ed. Guidelines for countermeasure of hepatitis A [in Japanese]. Tokyo: Viral Hepatitis Research Foundation of Japan, 1985~1-7. 15. Kark JD. Pre-exposure prophylaxis with immune serum globulin for prevention of viral hepatitis in army recruits. J Epidemiol Community Health 1982;36:176-182. 16. Kark JD. Pre-exposure prophylaxis of viral hepatitis with immune serum globulin in an endemic area. Scand J Infect Dis 1983; 15:3-6. 17. Lemon SM, Binn LN. Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983;148:1033-1039. 18. Stapleton JT, Jansen R, Lemon SM. Neutralizing antibody to hepatitis A virus in immune serum globulin and in the sera of human recipients of immune serum globulin. Gastroenterology 1985;89:637-642.
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I
To investigate the antibody titer necessary to inactivated adjuvant-free vaccine using the cell line prevent hepatitis A virus infection, either 15 or 7.5 GL37 and the HAV strain KRM003 (4). The vaccine mg/kg of immune serum globulin was injected into 10 consists of a suspension of 1 p,g/ml purified virus antihepatitis A virus negative volunteers and their (confirmed by ELISA) that is supplemented with preserum antihepatitis A virus titers were observed for 28 servatives and a vehicle before being freeze-dried. Phase wk. In addition, antibody titers were observed for 96 1 clinical trials started in July 1988 and have already wk in a phase 1 clinical trial of a hepatitis A vaccine. concluded. This vaccine has shown a good immunogeThe two studies were then compared to assess the immunogenicity of the vaccine and the persistence of nicity and produces a dose-dependent antibody response the antibody. Serum-neutralizing antibody titers that without side effects in humans (5). However, it was were greater than or equal to 4 (consideredas positive) difficult to conduct efficacy studies to determine the persisted for 18 wk and 14 wk after the injection of 15 minimum effective antibody titer necessary to prevent and 7.5 mg/kg of globulin, respectively. Hepatitis A HAV infection because no endemic areas for hepatitis A virus vaccine recipients showed adequate neutralizing were left in Japan. antibody titers, with the groups receiving 1, 0.5 and In this study, intramuscular ISG was given to 10 0.25 pg/dose showing titers of 46*5,44.7and 44, respec- anti-HAV-negative healthy male volunteers to predict tively, at 18 mo after the third inoculation. These the antibody titer necessary to preventing HAV infindings suggested that effective blood antibody titers were likely to be retained in the 1.0 kg or 0.5 Fg/dose fection. Their serum anti-HAV titers were observed for groups for at least several years. Moreover, the serum 28 wk. We also observed anti-HAV titers during the antihepatitis A virus titers demonstratedby a modified long-term follow-up of a group of hepatitis A vaccine radioimmunoassay changed in parallel with the neu- recipients. By comparing these two groups, the duration tralizing antibodytiters in the volunteers injectedwith of the effective antibody response and the immunogeglobulin. (HEPATOLOGY 1992;15983-988.) nicity of the vaccine were estimated.
Human immune serum globulin (ISG) has long been used prophylactically to prevent hepatitis A virus (HAV) infection, mainly for those people traveling abroad to areas where the virus is endemic or for close contacts of known hepatitis A cases (1).To improve the efficacy of ISG against hepatitis A, standard ISG preparations have been developed by the World Health Organization (WHO) (2). However, because the effective duration of the passive immunity afforded by ISG is limited by its serum half-life of 21 days or less (3) and because it is feared that the antibody concentration in ISG preparations may be decreasing, renewed calls have been heard for the development of a hepatitis A vaccine. In Japan, work has been undertaken to develop an Received July 10, 1991; accepted January 10, 1992. Address reprint requests to: Shigetoshi Fujiyama, M.D., Third Department of Internal Medicine, Kumamoto University Medical School, 1-1,Honjo 1-chome, Kumamoto 860, Japan. 31/1/36488
MATERIALS AND METHODS ?Globulin Trial. ?-Globulin (Lot No. 129-B) prepared by The Chemo-Sero-TherapeuticResearch Institute (Kumamoto, Japan) and containing 150 mg/ml of immune globulin was injected into the gluteus muscle in two groups of five male volunteers at a dose of 15 mgkg (group A) or 7.5 mgkg (group B). All the volunteers (mean age = 26.9 ? 3.0 yr, range = 23 to 34 yr) were negative for antibody to HAV. Blood samples were taken 1, 2, 3, 4, 5 and 7 days and 2, 3, 4, 6, 8, 10, 13, 16, 20, 24 and 28 wk after injection. Long-term Follow-up of Antibody Titers after Hepatitis A Vaccination. In the phase 1 clinical trial, hepatitis A vaccine
(Lot No. K-02, prepared by the above-mentionedinstitute) was injected intramuscularly into the upper arm in three groups of four healthy volunteers (mean age = 31.3 k 4.6 yr, range = 26 to 41 yr) who were negative for anti-HAV.The dose given was 0.25 ml (0.25 pg), 0.5 ml (0.5 pg) or 1.0 ml (1.0 pg), and the injection was performed at time zero and 1 and 6 mo later. The acquired anti-HAV titer was then measured at frequent intervals until the thirty-second week after the first injection. Subsequently, blood samples were collected at 48,72 and 96 wk to assess long-term antibody retention.
983
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FUJIYAMA ET AL.
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TABLE1. HAV antibody titers of human immune serum globulin Manufacturer
Lot No.
ELISA (mIUim1)"
N.T.b
HAVAB RIA"
Ad
918 919 920
121,000 129,000 120,000
1: 174,000 1:256,000 1: 195,000
1:800 1:663 1: 726
B'
050 052
30,000 33,000
1:64,000 1:48,000
1:154 1:217
Cf
126 127 129 131
155,000 118,000 126,000 139,000
1:182,000 1: 151,000 1: 169,000 1: 178,000
1: 721 1:800 1:668 1:711
WHO reference
No. F
100,000
1:600 (1: 442-1 : 762)
"Competitive inhibition ELISA. The antibody titer was calculated from the ratio of the 50% competitive inhibition titer for the simultaneously reacted positive control (2,000 mIU/ml) t o that of the test sample. bNeutralizing antibody titer (N.T.) was measured by ELISA using a GL37 monolayer cell sheet in a 96-well plate. 'Antibody titer was measured according to the procedure outlined for the HAVAB RIA kit (Abbott Laboratories, North Chicago, IL). dNihon Pharmaceutical Co. Ltd. (Tokyo, Japan). 'Japanese Red Cross (Tokyo, Japan). fThe Chemo-Sero-Therapeutic Research Institute (Kumamoto, Japan). gDev Biol Stand 1983;54:411-416.
Measurement of Anti-HAV Antibody Titers. Serum anti-HAV antibody titers were assessed by three of the following four methods. First was the competitive inhibition ELISA. The principle of this ELISA was similar to that of the HAVAB-EIA test (Abbott Laboratories, North Chicago, IL). Test samples were diluted with PBS (pH 7.2) containing 0.2% BSA and 0.05% Tween 20. Fourfold serial-diluted test samples and enzyme-labeled antibodies were reacted in antigen-coated plates. After washing, the enzyme substrate was added and color was developed. The antibody titer was calculated from the ratio of the 50% competitive inhibition titer for the simultaneously reacted positive control (2,000 mIU/ml, adjusted at the National Institutes of Health using WHO reference preparations) and that of the test sample. The second method was the neutralizing antibody assay. Neutralizing antibodies were measured by an ELISA using an HAV-sensitive GL37 monolayer cell sheet grown on a 96-well plate. Test samples were diluted with PBS (pH 7.2) containing 2% FBS and 0.002%Tween 80 (gentamicin was added at a final concentration of 50 pg/ml). Fourfold serial-diluted test samples and HAV at 50 to 100 median tissue culture infective doses (TCID,,)/ml were incubated at 37" C for 3 hr and then overnight at 4" C, after which each sample was added to four wells of the 96-well plate containing the GL37 monolayer and cultured. Two weeks later the cells were washed with PBS (pH 7.2) and fixed in 80% methanol supplemented with 30% hydrogen peroxide (1: 1,000). Then 50 pl of anti-HAV rabbit serum was added to each well, and the plates were incubated for 1 hr at 37" C. After further washing, 5Opl of horseradishconjugated goat antirabbit IgG (Medical and Biological Labs Co., Ltd., Nagoya, Japan) was added to each well, and the plates were incubated for 2 hr at 37" C. After final washing, the enzyme substrate was added and color was developed. Inhibition of more than 70% of the absorbance of the virus control was determined to be positive. The dilution that exhibited 50% neutralization was calculated by the method of Reed and Muench (6) and was defined as the neutralizing titer. The cutoff level was determined to be 4. The third method was anti-HAV RIA. Antibody titers were
measured according to the procedures outlined in the instructions of the HAVAJ3 RIA kit (Abbott Laboratories). This standard RIA was used to measure anti-HAV levels in the volunteers injected with vaccine. Commercial human ISG preparations were serially diluted from 1: 50 with PBS (pH 7.21, and the 50% competitive inhibition titer was determined. The fourth method was modified anti-HAV RIA. We also modified the RIA so that 0.1 ml of the test sample was used to compete with 0.1 ml of radiolabeled antibody observing the method of Provost et al. (7). This modified assay was used to measure anti-HAV levels in the volunteers injected with ISG. Informed consent was obtained from each volunteer before enrollment. This study was approved by the Ethics Committee of The Chemo-Sero-Therapeutic Research Institute.
RESULTS Anti-HAV Titers in Commercial Human ISG Preparations. Table 1summarizes the anti-HAV titers of the
globulin preparations commercially available in Japan, as determined by ELISA, neutralization and RIA. The WHO reference values obtained from the literature are also included. The globulin preparations studied consisted of a total of nine lots produced by three companies. Imported plasma was used by companies A and C , and domestic plasma obtained from blood donors was used by company B. The two lots from company B showed antibody titers that were approximately one fourth of those in the lots from companies A and C , regardless of the method of measurement used. The lots provided by companies A and C had titers exceeding those of the WHO reference preparations, whereas the lots from company B showed lower titers. Lot-129 from company C (used in this intramuscular globulin trial) gave an ELISA level of 126 IU/ml, a neutralizing titer of 1:169,000 and an RIA titer of 1:668. Comparison of the three measurement methods
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ANTI-HAV TITER AFTER ACTIVE OR PASSIVE IMMUNIZATION
showed that an anti-HAV titer of 1 IUiml corresponded to a neutralizing titer of 1:1,500 and a n RIA titer of 1:6. Time Course Changes of the Neutralizing Antibody Titer after Injection ofISG or Vaccination. The changes
in neutralizing antibody titers in the three vaccination groups and the two groups injected with ISG are shown in Figure 1. The half-life of the neutralizing antibody after ISG injection was 20 days in group A and 19 days in group B. Neutralizing titers above 4 (considered as positive) were respectively noted until 18and 14 wk after injection in groups A and B. Effective neutralizing antibody titers were achieved in the subjects inoculated with vaccine. In the groups receiving 1.0, 0.5 and 0.25 pgidose, the neutralizing antibody titers were respectively 43.9,44.0and 4'.O at 8 wk (1mo after the second inoculation); 43.7,43.2and 41.7 at 24 wk (5 mo after the second inoculation); 46.9, 45.9 and 45.4 at 28 wk (1 mo after the third inoculation); and 45.5,44.7and 44 at 96 wk (18 mo after the third inoculation). We compared the antibody neutralizing activity of serum obtained from vaccine recipients with that of the ISG preparations. Serum samples from vaccine recipients and samples of the ISG preparations were diluted and adjusted to ELISA values of 100, 10, 1 and 0.1 mIU/ml and then reacted with equal volumes of 102.6, 103.6 and 104.6 TCID,,/ml HAV suspensions. This mixture was then added to 8-well cell sheets, and the neutralizing activity was expressed as a percentage of the neutralizing-positive wells (Fig. 2). Serum samples were obtained 1mo after the third injection (28 wk) from subject No. 1of the 1.0 kg/dose group and subject No. 8 of the 0.5 pgidose group. All the serum samples from the two vaccine recipients and the two lots of ISG tested showed a similar HAV-neutralizing activity.
985
ISG
0 v1 v2
0
v3
24
48
72
96
weeks after the first injection
FIG. 1. Time course of neutralizing antibody titers in the three vaccinated groups ( 0 = 1.0 yg, = 0.5 yg, = 0.25 kg) and the two groups injected with ISG ( 0 = 15 mgkg, A = 7.5 mgkg).
and thus clinical efficacy trials and the determination of the minimum effective antibody titer are both difficult to perform. ISG has been used to prevent hepatitis A since 1945, and many reports have described its effectiveness (8-10). Direct proof of the efficacy of ISG was obtained in a human inoculation trial conducted by Krugman (11)in institutionalized children with mental retardation. However, among the various reports detailing the effectiveness of ISG in the prevention of hepatitis A, only a few have described the anti-HAV antibody titers of the ISG lots used. In a study of Japanese residents in Time Course of anti-HAV Titers after Iqjection of ISG Algeria, Arakawa et al. (12) used ISG lot Nos. 878, 883 or Vaccination a s Determined by RIA. The changes in and 884 from company A that had HAVAB RIA titers of the mean anti-HAV levels determined by RIA in the 1:400 to 1:800. The ISG preparation used in this study three vaccinated groups are shown in Figure 3. All the had a titer of 1:668 by RIA. Accordingly, the difference volunteers seroconverted to anti-HAV ( 2 50% inhi- in titers is not considered sufficient to influence the bition) at 1mo after the third vaccination, and an overall effectiveness of the preparations. Moreover, the titer of dose-dependence of the response was confirmed. The the ISG used exceeded that of the WHO reference changes in the mean anti-HAV levels determined by the preparations, which are adjusted t o ensure their effecmodified RIA are shown in Figure 4 for the two groups tiveness against HAV. The blood-neutralizing antibody injected with ISG. The mean inhibition value f 2 50%; titers of the subjects given ISG showed a half-life of 20 considered as positive) was retained for 11 and 8 wk, and 19 days, respectively, in the 15 and 7.5 mgkg respectively, in the 15 mgkg and 7.5 mgkg groups. groups, and these values are in close agreement with Titration of the anti-HAV titers in two different lots of those reported in the literature (3).If a neutralizing titer ISG using the standard and the modified RIAprocedures above 1:4 is considered positive, the duration of effective is shown in Figure 5 . The sensitivity of the modified antibody retention in the 15 and 7.5 mgkg groups was assay was about 10-fold greater than that of the 18 and 14 wk, respectively. These periods are similar to standard assay, and the 50% inhibition level in the the reported 2-mo duration of protection achieved with modified assay corresponded to an ELISA value of about a dose of 0.026 ml/kg (121, the 4-mo period achieved with 15 mIU/ml. a dose of 0.05 to 0.09 mVkg (13) and the 3-mo period achieved with the dose of 0.05 m l k g stipulated in the DISCUSSION Japanese guidelines for prophylaxis against hepatitis A Important issues to be considered in the development (14). of an inactivated hepatitis A vaccine are the appropriate Although many other studies of an even longer methods and criteria for evaluating its efficacy in duration of protection have been conducted (15, 16), humans. In Japan, no areas have been seen where those reports are difficult to evaluate because the large-scale outbreaks of hepatitis A are likely to occur, antibody titers of the ISG used were not stated.
HEPATOLOGY
FUJlYAMA ET AL.
986
ISG 127 100
10"TCID.Jml 10 '' TCIDr)rnl l o A 6TCIDdrnl
80 60 40
20
c
-
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1
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100
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.-
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1
Vaccine (No.1) T28 100
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* 0
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20
0) c
n
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01
1
10
100
Vaccine ( No.8 ) T28 100
80
60 40
20 0 01
1
10
100
HAV antibody titer (rnlUlrnl)
FIG.2. Neutralizing activity of the antibodies elicited by ISG and the vaccine. Two different lots of ISG (ISG 127 and ISG 131)were produced by company C. Two serum samples (No. 1 T28 and No. 8 T28) were obtained 1 mo after the third injection (28 wk)from subject No. 1 of the 1.0 pgldose group and subject No. 8 of the 0.5 pgldose group. A neutralization test to compare No. 8 T28 and HAV 102-6TCID,,/ml was not conducted.
Accordingly, in this study a neutralizing titer of 1:4 was taken to represent the antibody titer providing effective prophylaxis against hepatitis A, with a neutralizing titer of 1:4 corresponding to an ELISA result of 2 to 3 mIU/ml. Furthermore, in uztro cell culture tests showed that antibodies qualitatively equivalent to those found in ISG were produced when the vaccine was administered three times. Our findings indicated that the administration of the vaccine at 0.5 pg/dose or more would produce a high antibody titer for at least 5 mo after the second injection and for at least several years after the third injection (11, 17). Thus this study demonstrated that a third injection was probably effective in producing the long-term persistence of a protective antibody titer. One previous report, by Stapleton et al. (18),described the titers of neutralizing antibody to HAV in ISG preparations or in the serum of human ISG recipients. Because t,heir method of measurement differed from
ours, direct comparison of the two studies is difficult, but their ISG apparently possessed a lower antibody titer than that of the WHO reference preparations. They reported that when 18 healthy men had ISG administered at 0.02 mlkg all the serum samples collected on the third day and fifty fifth-day after injection contained detectable levels of neutralizing antibody. Both the report of Stapleton et al. (18) and our own results indicate that the neutralizing antibody titer provides good evidence of the value of ISG in the prophylaxis of hepatitis A and suggest that this test should also be used in evaluating the efficacy of hepatitis A vaccines. However, it is not a quick diagnostic test suitable for routine clinical use. We measured serum anti-HAV titers after vaccination by using the standard HAVAB RIA and assessed the titers after ISG injection by using a modified RIA. This modified assay appeared to permit about a 10-fold greater sensitivity of anti-HAV detection, a result that is
Vol. 15, No. 6, 1992 V l v2
v3
44
4
100 ,
0
'
0
'
'
'
987
ANTI-HAV TITER AFTER ACTIVE OR PASSIVE IMMUNIZATION
'
,
ISG
'
24
4,
100
,
,
,
,
,
48
.
'
.
'
,
T
'
72
,
'
,
4
0
,
12
8
96
20
16
24
28
weeks after the first injection
weeks after the first injection
FIG.3. Time course of HAV antibody titers in the three vaccination groups as determined by HAVAB RIA (0 = 1.0 pg, A = 0.5 p,g, 0 = 0.25 pg).
in agreement with the report of Provost et al. (7). In the present study, the 50% inhibition level in this modified RIA corresponded to an ELISA value of about 15 mIU/ml, indicating a n anti-HAV titer considerably higher than the 2 to 3 mIU/ml hypothesized to be necessary to prevent HAV infection. As compared with the standard assay, the modified assay had problems in terms of specificity. However, if the modified assay could be improved, its simplicity would allow its wider application in further vaccine studies. Measurement of the anti-HAV antibody titers in the ISG preparations sold by three different manufacturers demonstrated large differences related to the source of the plasma used. The antibody titers of the ISG made from imported plasma by two manufacturers were approximately the same and exceeded the anti-HAV antibody titers of the WHO reference preparations, whereas the titer of the ISG made from domestic blood donor plasma was lower than the WHO reference level and only one fourth of the titer in the other commercial ISG preparations. These results were not unexpected because the proportion of the population younger than 40 yr of age possessing anti-HAV is extremely low in Japan, and thus y-globulin preparations derived from Japanese blood donor plasma are likely to have low titers. Because Japan is tending to increasingly rely on domestic blood donor plasma as the sole source of all blood products, it is believed that a vaccine for the prevention of hepatitis A will become even more necessary in the future. The results of this study suggest that the recommended ISG dose for the prevention of hepatitis A should be decided on the basis of the anti-HAV antibody titer of each ISG lot and our data on the duration of effective antibody retention.
Acknowledgments: We wish to thank Ms. Mikiko Saito and Ms. Keiko Morozumi for their excellent technical assistance.
FIG.4. Time course of HAV antibody titers in the two groups injected with ISG as determined by the modified HAVAB RIA (0 = 15 mgikg, A = 7.5 mgkg).
100
1
t 0
h
e
6
I
A Q 0
0
I1 1
0
I
A 0
,
,
,
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,
.
,,....,
,
100
,
,
,...., 1000
,
,
, , , ,
J
10000
HAV antibody titer (mlUlml)
FIG.5. Titration of the hepatitis A antibody titer in two different lots of ISG (Lot 129 and Lot 131, which are represented respectively by triangles and circles) using the standard HAVAB RIA (open) and the modified HAVAB RIA (closed).
REFERENCES 1. Pollock TM, Reid D. Immunoglobulin for the prevention of infectious hepatitis in persons working overseas. Lancet 1969;1: 281-283. 2. Gerety RJ,SmallwoodLA, Finlayson JS, Tabor E. Standardization of the antibody to hepatitis A virus (anti-HAV) content of immunoglobulin. Dev Biol Stand 1983;54:411-416. 3. Wells JV.Metabolism of immunoglobulins. In: Fudenberg HH, Stites DP, Caldwell JL, Wells JV,eds. Basic and clinical immunology. Tokyo: Maruzen Co., 1976:195-203. 4. Moritsugu Y, Totsuka A. Hepatitis A vaccine: current status of development. In: Fukai K, ed. Virus vaccines in Asian countries. Tokyo: University of Tokyo Press, 1986:193-201. 5. Iino S, Fujiyama S, Horiuchi K, J o K. Clinical trial of inactivated hepatitis A vaccine [in Japanese]. Jpn J Gastroenterol 1990;87: 1532-1536. Muench H. A simple method of estimating fifty per cent 6. Reed U, endpoints. Am J Hyg 1938;27:493-497. 7. Provost PJ, Hughes JV,Miller WJ, Giesa PA, Banker FS, Emini EA. An inactivated heuatitis A viral vaccine of cell culture oripin. J Med Virol 1986;19:i3-31.
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8. Conrad ME, Lemon SM. Prevention of endemic icteric viral hepatitis by administration of immune serum gamma globulin. J Infect Dis 1987;156:56-63. 9. Green MS, Dotan K. Epidemiology: efficacy of immune serum globulin in an outbreak of hepatitis A virus infection in adults. J Infect 1988;17:265-270. 10. Green MS, Block C. Apparent effect of immune serum globulin prophylaxis in the military on viral hepatitis incidence in the civilian population in Israel. J Epidemiol Community Health 1989;43:187-190. 11. Krugman S. Effect of human immune serum globulin on infectivity of hepatitis A virus. J Infect Dis 1976;134:70-74. 12. Arakawa Y, Katsuhara N, Amaki S, Kaneda H, Honda T, Kanda Y, Satoh Y , et al. Assessment of human immune serum globulin in preventing hepatitis A infection among Japanese residents abroad [in Japanese]. Acta Hepatol Jap 1981;22:943-953. 13. Ohara H, Ebisawa I, Ohtani S. Prophylactic efficacy of immune serum globulin against hepatitis A. Jpn J Exp Med 1986;56: 229-233.
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14. Research Group of Hepatitis A. Countermeasure of prophylaxis. In: Ichida F, ed. Guidelines for countermeasure of hepatitis A [in Japanese]. Tokyo: Viral Hepatitis Research Foundation of Japan, 1985~1-7. 15. Kark JD. Pre-exposure prophylaxis with immune serum globulin for prevention of viral hepatitis in army recruits. J Epidemiol Community Health 1982;36:176-182. 16. Kark JD. Pre-exposure prophylaxis of viral hepatitis with immune serum globulin in an endemic area. Scand J Infect Dis 1983; 15:3-6. 17. Lemon SM, Binn LN. Serum neutralizing antibody response to hepatitis A virus. J Infect Dis 1983;148:1033-1039. 18. Stapleton JT, Jansen R, Lemon SM. Neutralizing antibody to hepatitis A virus in immune serum globulin and in the sera of human recipients of immune serum globulin. Gastroenterology 1985;89:637-642.
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