Clinical trial of a lyophilized inactivated hepatitis A candidate vaccine in healthy adult volunteers Shiro Iino*'**, Shigetoshi Fujiyama t, Kiyoshi Horiuchi ~;, Keiichiro Jyo ~, Yasushi Kuwabara ~, Seiya Sato ~, Shizuko Saika , Michio Morita ¶, Koichi Odohll Shoji Kuzuhara tl, Hiroaki Watanabe II, Masahiko Tanaka II and Kyosuke Mizuno II The safety and immunogenicity o f a lyophilized &activated hepatitis A vaccine was tested in healthy adult male volunteers. Thirty-six volunteers, all o f whom were negative f o r antibody to H A V (anti-HA V), were divided into three dosaoe 9roups, 1.0, 0.5 and 0.25 #9 o f viral protein, respectively. Each 9roup received a total course o f three intramuscular injections at months O, 1 and 6. Slight side effects were noted after 16 o f 99 injections and the occurrence and degree were almost identical to those o f other commercial vaccines. On the other hand, all subjects had measurable titres o f serum anti-HA V neutralizing antibodies as early as 2 months after the first injection. The mean values o f serum anti-HA V neutralizing antibodies at 7 months in the 1.0, 0.5 and 0.25 #g dose 9roups were 64-, 12-, and 9-fold higher, respectively, than those observed at 5 days in five recipients 9iven 7.5 my k9-1 body weight o f immune serum 91obulin ( ISG). Keywords:Hepatitis A; lyophilized; inactivated;vaccine;clinicaltrial
INTRODUCTION Hepatitis A is a common infection in most parts of the world, and the disease continues to be a significant problem in even the most highly industrialized countries. At present, only 1.5% of the population 30 years of age and younger in Japan is anti-HAV positive. The average annual incidence of infection is assumed to be approximately 100000 persons, and a high incidence is seen in the population between 20 and 40 years old. Administration of immunoglobulin has been the only specific prophylactic method for young people at high risk for hepatitis A. However, the use of immunoglobulin may not be practical for long-term prophylaxis, since the immunity lasts for only 4-6 months 1'2. The immunity acquired by hepatitis A vaccine is expected to be more potent and persistent. *First Department of Internal Medicine, Faculty of Medicine, Tokyo University, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo 113, Japan. tThird Department of Internal Medicine, Kumamoto University Medical School, 1-1, Honjo 1-chome, Kumamoto 860, Japan. tDepartment of Pediatrics, National East Tochigi Hospital, 2160 Shimo-okamoto, Kawachi, Tochigi 329-11, Japan. ~Denka Seiken Co. Ltd, 1-2-2 Minami-Honcho, Gosen, Niigata 959-16, Japan. ¶Chiba Serum Institute, 2-6-1, Konodai, Ichikawa, Chiba 272, Japan. liThe Chemo-Sero-Therapeutic Research Institute, Kyokushi, Kikuchi, Kumamoto 869-12, Japan. **To whom correspondence should be addressed. (Received 23 August 1991; revised 7 November 1991; accepted 8 November 1991) 0264-410X/92/050323-q~ © 1992Butterworth-HeinemannLtd
Feinstone et al. 3 visualized HAV particles for the first time in the stool of acutely infected patients using immune electron microscopy in 1973, and Provost et al. 4'5 established an animal model for HAV transmission, also in 1973, and succeeded in the isolation and propagation of HAV cell cultures in 1979. As a result, development of a vaccine has since advanced rapidly. At present, two kinds of vaccine, an inactivated vaccine6-8 and an attenuated live vaccine 9, are under development. However, the attenuated live vaccine must be further characterized before clinical trials can be performed 1°. We developed an inactivated adjuvant-free vaccine using an established cell line and HAV strain 11. A lyophilized vaccine was selected for the dose form of the inactivated vaccine based on the results of evaluation of immunogenicity and stability. We conducted a phase 1 study to evaluate the practical use of the lyophilized inactivated vaccine and consequently confirmed the superior safety and antibody induction capability. MATERIALS AND METHODS Cells and virus strain
While making a sensitivity index to HAV in cell-suspended fluid of the African green monkey kidney (lot 109) provided by the Japan Poliomyelitis Research Institute, a high-sensitivity cell strain, GL37, was established following serial passage and was cloned by Moritsugu et al. at the National Institute of Health 11. A
Vaccine, Vol. 10, Issue 5, 1992 323
Clinical trial of an HAV vaccine: S. lino et al.
virus strain was separated by Moritsugu et al. from the stool of a patient with sporadic hepatitis A infected in Fukuoka prefecture in 1979 and designated KRM00311. After this virus was passed 72 times through African green monkey kidney cell cultures, a virus strain for production was established following four times limit dilution passages in GL37. These cells and virus strain were supplied to the Chemo-Sero-Therapeutic Research Institute, Chiba Serum Institute, and Denka BioResearch Institute in Japan to conduct trial production of the vaccine.
a 92.5 k D a
VP-1 (33kDa)
Production of vaccine The virus product for vaccine preparation was made by inoculating HAV KRM003 with a multiplicity of infection of about 0.1 into GL37 cell-suspended fluid. The virus was incubated with the cells for 1 h at 37°C, and the infected cells were then cultured at 37°C for 1 week with Eagle's M E M supplemented with 8% fetal bovine serum (Bocknek Laboratories Inc., Canada). The concentration of serum was then reduced to 2% over 2 weeks. During the course, the culture fluid was exchanged every week. After washing the cultures in medium with phosphate-buffered saline (PBS), pH 7.2, to remove residual bovine serum, the HAV-infected cells were suspended with lysis buffer (1% N P 4 0 / 0 . 4 % D O C / 5 0 mM E D T A / 1 0 m M Tris-HC1 buffer, pH 7.5) and then harvested. Cell debris derived from the cells was removed by centrifugation at 8000 rev min -1 for 30 min. A polyethylene glycol 6000 (Wako Junyaku Co. Ltd, Japan) containing sodium chloride solution was added to the resultant supernatant at 1:4, the solution was stirred at 4°C for 2 h and was allowed to stand overnight. Then, the solution was centrifuged at 8000 rev m i n - 1 for 30 min and the resultant pellets were suspended with lysis buffer. The suspension was further centrifuged at 20000 rev min-1 overnight. The resultant virus pellets were resuspended in PBS, pH 7.2, and an equivalent volume of chloroform was added to the suspension to extract the virus at room temperature for 30 rain. DNase I (Takara Shuzo Co. Ltd, Japan) and RNase A (Sigma Chemical Co. Ltd, USA), whose final concentrations were 20-40 l~g ml- 1, respectively, and 50 pg m l - 1 of Proteinase K (Merck Co. Ltd, USA) were added to the aqueous phase. This enzyme treatment was continued for 5 h at 37°C. To this solution treated with the enzymes were added an equivalent volume of 2.5 M potassium phosphate buffer, pH 7.5, and 0.8 volume of a mixed solution ethoxyethanol and butoxyethanol (2:1 v/v ). By this organic solvent treatment, the virus was concentrated in the middle phase to form a band 12. The virus suspension obtained was centrifuged at 10 000 rev min 1 for 15 min and the resultant superantant was passed through a gel filtration column packed with Sephacryl S 400 Hr (Pharmacia, Sweden) using PBS, pH 7.2, containing 0.002% Tween 80 as an eluent buffer. Antigen-positive fractions were cozlected. To determine the purity of the purified HAV, protein and nucleic acid derived from host cells, and the residual amounts of detergents, enzymes and organic solvents derived from additives, were measured. The results demonstrated non-problematic residual levels, in which each component from the cells and additives was not detectable by any analysis method or was in very small amounts if at all present. For example, SDS-polyacrylamide
324 Vaccine, Vol. 10, Issue 5, 1992
66.2 kDa 45 kDa
31 kDa
VP0
21.5kDa
VP2 (31kDa) (31.5 kDa)
VP3 (29kDa)
b
J
Figure 1 (a) SDS-polyacrylamide gel electrophoresis and (b) densitometry of the hepatitis A vaccine preparation
gel electrophoresis and densitometry of purified HAV are shown in Figure 1. There was no protein detected except for the specific viral protein (33, 31.5, 31 and 29 kDa ). We measured the amount of protein derived from host cells and bovine serum using a dot-blot assay. There was < 1.9% of residual protein in the purified HAV. Next, we measured the amount of nucleic acid derived from host cells using DNA-hybridization. There was less than 5 pg of residual nucleic acid in 0.5/~g of purified HAV. The highly purified virus was inactivated with formalin at 1:4000 at 37°C for 12 days. Inactivation was confirmed by sampling at days 8 and 12, and inoculation to GL37 cells demonstrating no trace of virus growth using the immunofocus method. After confirmation of inactivation, the immunogenicity and efficacy of both the full virion and the empty virion of GL37-derived HAV were investigated by Moritsugu et al. using marmosets. It was shown that both full virion and empty virion of GL37-derived HAV effectively provided protection against hepatitis A virus infection. We then studied the dose form of the inactivated vaccine. Table 1 shows that the relative potency of freeze-dried vaccine is equal to that of aqueous vaccine. Based on this relative potency test and the stability of the purified HAV, we selected the lyophilized vaccine as the dose form of the inactivated vaccine. The inactivated solution was suspended with phosphate-buffered saline containing stabilizer to give 1.0 pg m1-1 antigen and was then subjected to fereeze-drying in a small container. This was subsequently used for the phase 1 study. We also conducted various tests for trial vaccines according to
Clinical trial of an HAV vaccine: S. lino et al.
the guidelines of the Pharmacopoeia of the Ministry of Health and Welfare of Japan, and achieved satisfactory results required by the guidelines in all tests. The vaccine used was developed by the Chemo-Sero-Therapeutic Research Institute (lot K-02), Denka Bio-Research Institute (lot D-01 ) and Chiba Serum Institute (lot C-01 ) under the three-way collaboration. Identity of the three vaccine lots used was proved by the evaluation of purity and amount of HAV antigen at the National Institute of Health.
Detection of total anti-HAV by ELISA
Detection of neutralizing antibodies to HAV
Detection of total and IgM class anti-HAV by radioimmunoassay
The principle of ELISA was similar to that of the HAVAB-EIA test (Abbott Laboratories, North Chicago, IL). Diluted test samples and enzyme-labelled antibodies were reacted in antigen-coated plates. After washing, the enzyme substrate was added and colour was developed. The antibody titre was calculated from the ratio of the 50% competitive inhibition titre for the simultaneously reacted positive control at 2000 mIU ml-1 (adjusted at the National Institute of Health using a WHO reference preparation 14) and that of the test sample.
Neutralizing antibodies (NT) were measured by ELISA using a GL37 monolayer cell sheet of a 96-well plate. For the determination, diluted sample serum was reacted with HAV of 50-100 TCIDso for 3 h at 37°C and subsequently at 4°C overnight and then inoculated into each four wells on the GL37 monolayer cell sheet of the 96-well plate. A well inhibition of > 70% of the absorbance of virus control was determined to be positive. The dilution which exhibited 50% neutralization was calculated by the Reed-Miiench method 13 and was defined as the neutralizing titre. The cut-off level was determined to be 41 .
Total anti-HAV antibodies were measured following procedures outlined in the HAVAB RIA kit directions (Abbott Laboratories, North Chicago, IL) and expressed as percentage inhibition. Those sera which inhibited binding by > 50% were considered positive. IgM class anti-HAV antibodies were measured following procedures outlined in the HAVAB-M RIA kit directions (Abbott Laboratories) and expressed in the cut-off index. Those sera which showed cut-off index values of ~>1 were considered positive. Volunteers Healthy adult males, HAV antibody negative, who understood the study of HAV vaccine and had given their informed consent to the content, were the subjects.
Table I Relative potency of freeze-dried and absorbed vaccines versus aqueous vaccine Relative potency in
Aqueous vaccine Freeze-dried vaccine Alum-absorbed vaccine
ddY mouse
SD rat
Mean value
1.000 1.234 2.420
1.000 0.867 1.790
1.000 1.051 2.105
Vaccination and examination Thirty-six volunteers were allocated into three groups of 12 persons by lot. Each group of 12 volunteers was then randomly subdivided into three dose-groups. Each group received an injection into the triceps muscle with 0.25, 0.5 or 1.0 #g of vaccine, a total of three times, at weeks 0, 4, and 24. As shown in Figure 2, physical findings were evaluated, and blood, urine and faecal samples were collected for analysis. Examinations of peripheral blood, usual serum biochemistry, and HAV antibody were investigated.
Using physiological saline as diluent, vaccines were diluted exactly to 400, 200, 100, 50 and 25 ngHAV Ag m1-1. Eight ddY mice or five SD rats of 5 weeks of age were provided for each dilution, and each animal received 1 ml intraperitoneal injection. The animals were bled 5 weeks later, and examined for 50% inhibition anti-HAV titres by quantitative ELISA. Relative potencies were then calculated using the parallel line assay method
Months
I
2
3
4
6
7
8
24
28
32
I
I
Weeks
0
4
8
12
16
Day
J, I , ,
i , , , ,
J
J
I
t
t
•
•
00
00
Vaccination
II,
,
,
t
NT assay
Blood count Blood chemical exam.
Urine exam.
Isolation of HAV
/
O•
00000
Physical exam.
Figure 2
Schedule of the inactivated hepatitis A vaccine trial
Vaccine, Vol. 10, Issue 5, 1992
325
Clinical trial of an HAV vaccine: S. linD et al.
ISG injection trial In order to compare the immune response acquired by the vaccine with that acquired by ISG, five seronegative male volunteers (mean age 27 + 2 years, range 25-30 years) were given 7.5 mg kg- 1 body weight of ISG (gamma globulin lot 129-B, The Chemo-SeroTherapeutic Research Institute) containing 150 mg ml-1 immune globulin. Serum samples were obtained 5 days after injection. RESULTS Thirty-three of the 36 volunteers were analysed. One volunteer of the group with 0.25 #g, one with 1.0 #g and one with 0.5 #g were excluded, respectively, since the third injection could not be conducted because of a common cold in the first two people and varicella in the third person. Table 2 shows the age distribution of the volunteers. The mean age of the 33 volunteers was 31.1 + 7.8 years. Mean ages of the volunteers for vaccine lots D-01, C-01 and K-02 were 26.2 __+2.2, 34.8 _+ 10.7 and 31.3 + 4.6 years, respectively. Table 3 shows the frequency of side effects developed by the volunteers. The reported number of side effects declined with increasing number Of injections. The frequency of side effects was not affected by the injection dose. The main side effect was localized pain at the injection site. Ten of 99 injections gave rise to localized pain, although this side effect was observed only on the injection day and the following day and was mild. Generalized reactions, which included three cases of malaise and one case of drowsiness, were also mild. The frequencies of side effects for vaccine lots D-01, C-01 and K-02 were 14.8, 13.9 and 19.4%, respectively. Accordingly, a large difference in frequencies of side effects for the three vaccine lots was not shown. Examinations were conducted according to the schedule shown in Figure 2. Abnormal levels suspected
to be due to injection of vaccine, especially that of hepatitis, were not recognized on urinary analysis (four items), peripheral blood examinations (seven items), or haematological and biochemical examinations (20 items). Isolation of HAV was attempted, but HAV was not detected. Figure 3 shows the time course of the geometric mean NT titre in the three dose-groups with injection of the vaccine. As a whole, dose response was confirmed. At 2 months after the first injection, all cases were positive. After the third injection, the geometric mean NT titre increased dramatically in all groups. At 1 month after the third injection, the geometric mean NT titres of the 1.0 #g dose-group for vaccine lots D-01, C-01 and K-02 were 46'2, 46.6 and 469, respectively. A large difference in the geometric mean NT titres for the three vaccine lots was not shown. On the other hand, the geometric mean NT titre of the subjects given ISG in the 7.5 mg kg- 1 group was 43.6 at 5 days after injection. All persons given HAV vaccine had serum NT titres of >/43.6 at 1 month after the third injection. Figure 4 shows the time course of the geometric mean total anti-HAV titre determined by ELISA in the three dose-groups with injection of the vaccine. The geometric mean total anti-HAV titres at 2 months after the first injection in the 1.0, 0.5 and 0.25 #g dose-groups were 562, 323 and 134 mlU m1-1 respectively, and at 7 months, 3630, 1288 and 1000 mlU ml-1, respectively. The changes in mean HAVAB RIA titres in the three dose-groups of vaccinees injected with lot K-02 are shown in Figure 5. All vaccinees had ~>50% competitive inhibition titres at 1 month after the third injection. The changes in mean HAVAB-M RIA titres in the three dose-groups of vaccinees injected with lot K-02
Vl
+~
Table
2
0.5/~g
0-19 20 - 29 30-39 40 49 />50
1 7 2 1 -
6 3 2
Mean age (years)
28.7_+7.5
30.3+_5.4
q7 _
o c
Dosage group 1.0/~g
V3
>. -(3
Age distribution of volunteers
Age (years)
V2
0.25 #g
Total
4 4 2 1
1 17 9 5 1
34.4_+9.0
31.1 _+7.8
45
43
Z
INTRODUCTION Hepatitis A is a common infection in most parts of the world, and the disease continues to be a significant problem in even the most highly industrialized countries. At present, only 1.5% of the population 30 years of age and younger in Japan is anti-HAV positive. The average annual incidence of infection is assumed to be approximately 100000 persons, and a high incidence is seen in the population between 20 and 40 years old. Administration of immunoglobulin has been the only specific prophylactic method for young people at high risk for hepatitis A. However, the use of immunoglobulin may not be practical for long-term prophylaxis, since the immunity lasts for only 4-6 months 1'2. The immunity acquired by hepatitis A vaccine is expected to be more potent and persistent. *First Department of Internal Medicine, Faculty of Medicine, Tokyo University, 3-1, Hongo 7-chome, Bunkyo-ku, Tokyo 113, Japan. tThird Department of Internal Medicine, Kumamoto University Medical School, 1-1, Honjo 1-chome, Kumamoto 860, Japan. tDepartment of Pediatrics, National East Tochigi Hospital, 2160 Shimo-okamoto, Kawachi, Tochigi 329-11, Japan. ~Denka Seiken Co. Ltd, 1-2-2 Minami-Honcho, Gosen, Niigata 959-16, Japan. ¶Chiba Serum Institute, 2-6-1, Konodai, Ichikawa, Chiba 272, Japan. liThe Chemo-Sero-Therapeutic Research Institute, Kyokushi, Kikuchi, Kumamoto 869-12, Japan. **To whom correspondence should be addressed. (Received 23 August 1991; revised 7 November 1991; accepted 8 November 1991) 0264-410X/92/050323-q~ © 1992Butterworth-HeinemannLtd
Feinstone et al. 3 visualized HAV particles for the first time in the stool of acutely infected patients using immune electron microscopy in 1973, and Provost et al. 4'5 established an animal model for HAV transmission, also in 1973, and succeeded in the isolation and propagation of HAV cell cultures in 1979. As a result, development of a vaccine has since advanced rapidly. At present, two kinds of vaccine, an inactivated vaccine6-8 and an attenuated live vaccine 9, are under development. However, the attenuated live vaccine must be further characterized before clinical trials can be performed 1°. We developed an inactivated adjuvant-free vaccine using an established cell line and HAV strain 11. A lyophilized vaccine was selected for the dose form of the inactivated vaccine based on the results of evaluation of immunogenicity and stability. We conducted a phase 1 study to evaluate the practical use of the lyophilized inactivated vaccine and consequently confirmed the superior safety and antibody induction capability. MATERIALS AND METHODS Cells and virus strain
While making a sensitivity index to HAV in cell-suspended fluid of the African green monkey kidney (lot 109) provided by the Japan Poliomyelitis Research Institute, a high-sensitivity cell strain, GL37, was established following serial passage and was cloned by Moritsugu et al. at the National Institute of Health 11. A
Vaccine, Vol. 10, Issue 5, 1992 323
Clinical trial of an HAV vaccine: S. lino et al.
virus strain was separated by Moritsugu et al. from the stool of a patient with sporadic hepatitis A infected in Fukuoka prefecture in 1979 and designated KRM00311. After this virus was passed 72 times through African green monkey kidney cell cultures, a virus strain for production was established following four times limit dilution passages in GL37. These cells and virus strain were supplied to the Chemo-Sero-Therapeutic Research Institute, Chiba Serum Institute, and Denka BioResearch Institute in Japan to conduct trial production of the vaccine.
a 92.5 k D a
VP-1 (33kDa)
Production of vaccine The virus product for vaccine preparation was made by inoculating HAV KRM003 with a multiplicity of infection of about 0.1 into GL37 cell-suspended fluid. The virus was incubated with the cells for 1 h at 37°C, and the infected cells were then cultured at 37°C for 1 week with Eagle's M E M supplemented with 8% fetal bovine serum (Bocknek Laboratories Inc., Canada). The concentration of serum was then reduced to 2% over 2 weeks. During the course, the culture fluid was exchanged every week. After washing the cultures in medium with phosphate-buffered saline (PBS), pH 7.2, to remove residual bovine serum, the HAV-infected cells were suspended with lysis buffer (1% N P 4 0 / 0 . 4 % D O C / 5 0 mM E D T A / 1 0 m M Tris-HC1 buffer, pH 7.5) and then harvested. Cell debris derived from the cells was removed by centrifugation at 8000 rev min -1 for 30 min. A polyethylene glycol 6000 (Wako Junyaku Co. Ltd, Japan) containing sodium chloride solution was added to the resultant supernatant at 1:4, the solution was stirred at 4°C for 2 h and was allowed to stand overnight. Then, the solution was centrifuged at 8000 rev m i n - 1 for 30 min and the resultant pellets were suspended with lysis buffer. The suspension was further centrifuged at 20000 rev min-1 overnight. The resultant virus pellets were resuspended in PBS, pH 7.2, and an equivalent volume of chloroform was added to the suspension to extract the virus at room temperature for 30 rain. DNase I (Takara Shuzo Co. Ltd, Japan) and RNase A (Sigma Chemical Co. Ltd, USA), whose final concentrations were 20-40 l~g ml- 1, respectively, and 50 pg m l - 1 of Proteinase K (Merck Co. Ltd, USA) were added to the aqueous phase. This enzyme treatment was continued for 5 h at 37°C. To this solution treated with the enzymes were added an equivalent volume of 2.5 M potassium phosphate buffer, pH 7.5, and 0.8 volume of a mixed solution ethoxyethanol and butoxyethanol (2:1 v/v ). By this organic solvent treatment, the virus was concentrated in the middle phase to form a band 12. The virus suspension obtained was centrifuged at 10 000 rev min 1 for 15 min and the resultant superantant was passed through a gel filtration column packed with Sephacryl S 400 Hr (Pharmacia, Sweden) using PBS, pH 7.2, containing 0.002% Tween 80 as an eluent buffer. Antigen-positive fractions were cozlected. To determine the purity of the purified HAV, protein and nucleic acid derived from host cells, and the residual amounts of detergents, enzymes and organic solvents derived from additives, were measured. The results demonstrated non-problematic residual levels, in which each component from the cells and additives was not detectable by any analysis method or was in very small amounts if at all present. For example, SDS-polyacrylamide
324 Vaccine, Vol. 10, Issue 5, 1992
66.2 kDa 45 kDa
31 kDa
VP0
21.5kDa
VP2 (31kDa) (31.5 kDa)
VP3 (29kDa)
b
J
Figure 1 (a) SDS-polyacrylamide gel electrophoresis and (b) densitometry of the hepatitis A vaccine preparation
gel electrophoresis and densitometry of purified HAV are shown in Figure 1. There was no protein detected except for the specific viral protein (33, 31.5, 31 and 29 kDa ). We measured the amount of protein derived from host cells and bovine serum using a dot-blot assay. There was < 1.9% of residual protein in the purified HAV. Next, we measured the amount of nucleic acid derived from host cells using DNA-hybridization. There was less than 5 pg of residual nucleic acid in 0.5/~g of purified HAV. The highly purified virus was inactivated with formalin at 1:4000 at 37°C for 12 days. Inactivation was confirmed by sampling at days 8 and 12, and inoculation to GL37 cells demonstrating no trace of virus growth using the immunofocus method. After confirmation of inactivation, the immunogenicity and efficacy of both the full virion and the empty virion of GL37-derived HAV were investigated by Moritsugu et al. using marmosets. It was shown that both full virion and empty virion of GL37-derived HAV effectively provided protection against hepatitis A virus infection. We then studied the dose form of the inactivated vaccine. Table 1 shows that the relative potency of freeze-dried vaccine is equal to that of aqueous vaccine. Based on this relative potency test and the stability of the purified HAV, we selected the lyophilized vaccine as the dose form of the inactivated vaccine. The inactivated solution was suspended with phosphate-buffered saline containing stabilizer to give 1.0 pg m1-1 antigen and was then subjected to fereeze-drying in a small container. This was subsequently used for the phase 1 study. We also conducted various tests for trial vaccines according to
Clinical trial of an HAV vaccine: S. lino et al.
the guidelines of the Pharmacopoeia of the Ministry of Health and Welfare of Japan, and achieved satisfactory results required by the guidelines in all tests. The vaccine used was developed by the Chemo-Sero-Therapeutic Research Institute (lot K-02), Denka Bio-Research Institute (lot D-01 ) and Chiba Serum Institute (lot C-01 ) under the three-way collaboration. Identity of the three vaccine lots used was proved by the evaluation of purity and amount of HAV antigen at the National Institute of Health.
Detection of total anti-HAV by ELISA
Detection of neutralizing antibodies to HAV
Detection of total and IgM class anti-HAV by radioimmunoassay
The principle of ELISA was similar to that of the HAVAB-EIA test (Abbott Laboratories, North Chicago, IL). Diluted test samples and enzyme-labelled antibodies were reacted in antigen-coated plates. After washing, the enzyme substrate was added and colour was developed. The antibody titre was calculated from the ratio of the 50% competitive inhibition titre for the simultaneously reacted positive control at 2000 mIU ml-1 (adjusted at the National Institute of Health using a WHO reference preparation 14) and that of the test sample.
Neutralizing antibodies (NT) were measured by ELISA using a GL37 monolayer cell sheet of a 96-well plate. For the determination, diluted sample serum was reacted with HAV of 50-100 TCIDso for 3 h at 37°C and subsequently at 4°C overnight and then inoculated into each four wells on the GL37 monolayer cell sheet of the 96-well plate. A well inhibition of > 70% of the absorbance of virus control was determined to be positive. The dilution which exhibited 50% neutralization was calculated by the Reed-Miiench method 13 and was defined as the neutralizing titre. The cut-off level was determined to be 41 .
Total anti-HAV antibodies were measured following procedures outlined in the HAVAB RIA kit directions (Abbott Laboratories, North Chicago, IL) and expressed as percentage inhibition. Those sera which inhibited binding by > 50% were considered positive. IgM class anti-HAV antibodies were measured following procedures outlined in the HAVAB-M RIA kit directions (Abbott Laboratories) and expressed in the cut-off index. Those sera which showed cut-off index values of ~>1 were considered positive. Volunteers Healthy adult males, HAV antibody negative, who understood the study of HAV vaccine and had given their informed consent to the content, were the subjects.
Table I Relative potency of freeze-dried and absorbed vaccines versus aqueous vaccine Relative potency in
Aqueous vaccine Freeze-dried vaccine Alum-absorbed vaccine
ddY mouse
SD rat
Mean value
1.000 1.234 2.420
1.000 0.867 1.790
1.000 1.051 2.105
Vaccination and examination Thirty-six volunteers were allocated into three groups of 12 persons by lot. Each group of 12 volunteers was then randomly subdivided into three dose-groups. Each group received an injection into the triceps muscle with 0.25, 0.5 or 1.0 #g of vaccine, a total of three times, at weeks 0, 4, and 24. As shown in Figure 2, physical findings were evaluated, and blood, urine and faecal samples were collected for analysis. Examinations of peripheral blood, usual serum biochemistry, and HAV antibody were investigated.
Using physiological saline as diluent, vaccines were diluted exactly to 400, 200, 100, 50 and 25 ngHAV Ag m1-1. Eight ddY mice or five SD rats of 5 weeks of age were provided for each dilution, and each animal received 1 ml intraperitoneal injection. The animals were bled 5 weeks later, and examined for 50% inhibition anti-HAV titres by quantitative ELISA. Relative potencies were then calculated using the parallel line assay method
Months
I
2
3
4
6
7
8
24
28
32
I
I
Weeks
0
4
8
12
16
Day
J, I , ,
i , , , ,
J
J
I
t
t
•
•
00
00
Vaccination
II,
,
,
t
NT assay
Blood count Blood chemical exam.
Urine exam.
Isolation of HAV
/
O•
00000
Physical exam.
Figure 2
Schedule of the inactivated hepatitis A vaccine trial
Vaccine, Vol. 10, Issue 5, 1992
325
Clinical trial of an HAV vaccine: S. linD et al.
ISG injection trial In order to compare the immune response acquired by the vaccine with that acquired by ISG, five seronegative male volunteers (mean age 27 + 2 years, range 25-30 years) were given 7.5 mg kg- 1 body weight of ISG (gamma globulin lot 129-B, The Chemo-SeroTherapeutic Research Institute) containing 150 mg ml-1 immune globulin. Serum samples were obtained 5 days after injection. RESULTS Thirty-three of the 36 volunteers were analysed. One volunteer of the group with 0.25 #g, one with 1.0 #g and one with 0.5 #g were excluded, respectively, since the third injection could not be conducted because of a common cold in the first two people and varicella in the third person. Table 2 shows the age distribution of the volunteers. The mean age of the 33 volunteers was 31.1 + 7.8 years. Mean ages of the volunteers for vaccine lots D-01, C-01 and K-02 were 26.2 __+2.2, 34.8 _+ 10.7 and 31.3 + 4.6 years, respectively. Table 3 shows the frequency of side effects developed by the volunteers. The reported number of side effects declined with increasing number Of injections. The frequency of side effects was not affected by the injection dose. The main side effect was localized pain at the injection site. Ten of 99 injections gave rise to localized pain, although this side effect was observed only on the injection day and the following day and was mild. Generalized reactions, which included three cases of malaise and one case of drowsiness, were also mild. The frequencies of side effects for vaccine lots D-01, C-01 and K-02 were 14.8, 13.9 and 19.4%, respectively. Accordingly, a large difference in frequencies of side effects for the three vaccine lots was not shown. Examinations were conducted according to the schedule shown in Figure 2. Abnormal levels suspected
to be due to injection of vaccine, especially that of hepatitis, were not recognized on urinary analysis (four items), peripheral blood examinations (seven items), or haematological and biochemical examinations (20 items). Isolation of HAV was attempted, but HAV was not detected. Figure 3 shows the time course of the geometric mean NT titre in the three dose-groups with injection of the vaccine. As a whole, dose response was confirmed. At 2 months after the first injection, all cases were positive. After the third injection, the geometric mean NT titre increased dramatically in all groups. At 1 month after the third injection, the geometric mean NT titres of the 1.0 #g dose-group for vaccine lots D-01, C-01 and K-02 were 46'2, 46.6 and 469, respectively. A large difference in the geometric mean NT titres for the three vaccine lots was not shown. On the other hand, the geometric mean NT titre of the subjects given ISG in the 7.5 mg kg- 1 group was 43.6 at 5 days after injection. All persons given HAV vaccine had serum NT titres of >/43.6 at 1 month after the third injection. Figure 4 shows the time course of the geometric mean total anti-HAV titre determined by ELISA in the three dose-groups with injection of the vaccine. The geometric mean total anti-HAV titres at 2 months after the first injection in the 1.0, 0.5 and 0.25 #g dose-groups were 562, 323 and 134 mlU m1-1 respectively, and at 7 months, 3630, 1288 and 1000 mlU ml-1, respectively. The changes in mean HAVAB RIA titres in the three dose-groups of vaccinees injected with lot K-02 are shown in Figure 5. All vaccinees had ~>50% competitive inhibition titres at 1 month after the third injection. The changes in mean HAVAB-M RIA titres in the three dose-groups of vaccinees injected with lot K-02
Vl
+~
Table
2
0.5/~g
0-19 20 - 29 30-39 40 49 />50
1 7 2 1 -
6 3 2
Mean age (years)
28.7_+7.5
30.3+_5.4
q7 _
o c
Dosage group 1.0/~g
V3
>. -(3
Age distribution of volunteers
Age (years)
V2
0.25 #g
Total
4 4 2 1
1 17 9 5 1
34.4_+9.0
31.1 _+7.8
45
43
Z
