Safety and immunogenicity of an inactivated hepatitis A candidate vaccine in healthy adult volunteers G. Wiedermann *§, F. Ambroseh*,:H. Kollaritsch*, H. Hofmann t, Ch. Kunz f, E. D'Hondt ~, A. Delem ~, F.E. AnilrC, A. Safary ~ and J. St6phenne ~ The reactogenicity and immunogenicity o f a formaldehyde-inactivated hepatitis A vaccine have been investigated. Three different dose levels o f vaccine (180, 360 and 720 E L I S A units) were administered to healthy volunteers according to a O, 1, 2 and 12 month schedule. The vaccine was safe and well tolerated. Reactions observed following vaccination were essentially mild and were not dependent upon the quantity of antigen administered. All subjects had measurable titres of anti-HA V antibodies after the full vaccination course; the immune response to the vaccine was dose-related. Antibody titres in vaccinees at month 13 were between 60- and 190-fold higher than those observed in a group o f subjects given anti-HA V immunoglobulin.
Keywords:Candidate vaccine; hepatitis A; ELISA; inactivated
Introduction
Subjects and methods
Hepatitis A is a c o m m o n infectious disease present throughout the world. It is more prevalent in areas of poor sanitary conditions and low socioeconomic levels although sporadic outbreaks have been shown to occur in developed countries 1. Male homosexuals and intravenous drug addicts have been shown to be at increased risk of infection 2'3. Rates of infection are high in institutions such as pre-school day-care centres, prisons and mental hospitals where large groups of people are present under crowded conditions 3-5. Travellers to developing countries and military personnel are also at-risk groups 6-a. Although, unlike hepatitis B, hepatitis A does not lead to chronic infection, it is nevertheless an important cause of morbidity. At present, the only means of specific prophylaxis is passive immunization with immunoglobulins (ISG). The protection afforded by ISG is, however, transient 9. For long-term protection of high-risk groups, a safe and effective vaccine would be the most cost beneficial. In 1979, Provost and Hilleman 1° showed that the hepatitis A virus could be maintained in culture, permitting the development of both live, attenuated 11-13 and inactivated 14-17 candidate vaccines. The results of a clinical study in humans with a formaldehydeinactivated, whole-virion vaccine are reported here.
Trial p r o t o c o l A group of 110 healthy volunteers without antibodies against the hepatitis A virus initially gave their consent to participate in this study. They were allocated into three groups, the first two being randomized. Three subjects were found to have been infected with hepatitis B and were therefore excluded from the study. Two volunteers received vaccine at 0, 2 and 3 months, one other failed to return after one dose of vaccine; these subjects are not included in the analysis. The remaining volunteers were aged between 17 and 55 years (average of 26.7 years). The ratio of males to females was 0.93. The vaccine was administered in the deltoid muscle following a 0, 1, 2 month vaccination schedule. An additional dose of vaccine was given at month 12. Each dose contained either 180, 360 or 720 ELISA units (EU, see below) of killed hepatitis A virus. Blood samples for antibody determination were drawn before, and 1 month after, each vaccine.dose. In order to compare the immune response elicited by the candidate vaccine with anti-HAV titres following passive immunization, 23 seronegative volunteers (mean age 23.1 years; ratio of males to females, 0.83) were given 100IU per 60kg body weight of ISG (Globuman Hepatitis A Berna, Swiss Serum and Vaccine Institute). Serum samples were obtained 5 days later and anti-HAV titres evaluated by ELISA. Vaccine safety and reactogenicity were assessed by means of standardized checklists, where local, general or 'other' signs and symptoms were recorded on a four point, absent to severe, scale on the day of vaccination and for 3 days after each vaccine dose. Serum levels of liver-specific enzymes (aspartate aminotransferase, alanine aminotransferase, y-glutamyltranspeptidase and alkaline phosphatase) and serum bilirubin concentrations were also measured before and after vaccination.
*Institute of Specific Prophylaxis and Tropical Medicine, University of Vienna, Austria. t Institute of Virology, University of Vienna, Austria. tSmithKline Biologicals, Rixensart, Belgium. §To whom correspondence should be addressed at: Institute for Spezifische Prophylaxe und Tropenmedizin der Universit&t, Kinderspitalgasse 15, 1095 Vienna, Austria. (Received 30 April 1990; accepted 11 June 1990) 0264-410X/90/060581---04 © 1990 Butterworth-HeinernannLtd
Vaccine, Vol. 8, December 1990 581
Inactivated hepatitis A candidate vaccine: G. Wiedermann et al.
The study was conducted in accordance with the provisions of the Declaration of Helsinki. The trial was approved by the ethical commission of the University of Vienna. Written informed consent was obtained from a l l volunteers before vaccination.
Table 1 Percentages of vaccinees with or without symptoms following vaccination with 360 EU of the hepatitis A candidate vaccine Subjects with symptoms
Subjects Vaccine
Number of
dose
documented
Vaccine
number
injections"
The vaccine was prepared from the C L F strain (RIT 4379) grown on MRC-5 cells. The virus was purified by ultrafiltration and chromatography and inactivated with formaldehyde, as described previously 17. The vaccine meets the requirements of the W H O and the US Food and Drug Administration for similar inactivated vaccines~ 8,~9. The antigen content of the vaccine was measured by means of a capture ELISA (using purified peroxidase labelled IgG conjugate, see Ref. 17). Three different doses with 180, 360 and 720EU, absorbed onto 0.5mg of AI(OH)3 were prepared.
1
Serology Liver enzyme activities and serum bilirubin levels were measured using standard laboratory techniques. Antibodies against HAV were measured using an ELISA inhibition assay. Titres were calculated in mlU ml-1 in comparison with a standardized immunoglobulin preparation (obtained from the WHO) using the four parameter method 2°. Sera with titres below 20 mlU ml-1 were considered negative. Anti-HAV were also quantified using a commercially available radioimmunoassay (RIA; HAVAB, Abbott Laboratories, North Chicago, IL) and expressed in percentage inhibition. In accordance with the manufacturer's instructions, only those sera which inhibited binding by more than 50% were taken as positive. Neutralizing antibodies were assayed using the radioimmunofocus inhibition test (RIFIT, Ref. 21) on MRC-5 cells, using serial dilutions of serum (starting at 1/40 or 1/80 as indicated). RIFIT titres are expressed as the reciprocal of the highest serum dilution neutralizing 50% of the HAV foci. Purified virus.was prepared as described previously 22.
Statist&al analysis z 2 analysis and Student's t test, as indicated, were used to compare results. Statistical comparisons on the immune response in subjects administered 720 EU were not performed as this group was vaccinated separately at the end of the study.
Results Evaluation of vaccine safety and reactogenlcity Liver function tests to evaluate any eventual vaccineinduced hepatocellular damage were performed on serum samples from volunteers throughout the vaccination course. No clinically significant long-term increases in the activities of the four enzymes tested nor serum bilirubin concentrations were evidenced. In general, the frequency of adverse reactions decreased with successive vaccine doses, indicating that the candidate vaccine did not induce hypersensitivity reactions even after the administration of four doses. The results for volunteers given 360 EU doses are shown in
Table I. The effect of dose level on vaccine reactogenicity is
582
Vaccine, Vol. 8, D e c e m b e r 1990
Local only (%)
General only (%)
Local and general (%)
without symptoms
49
27 (55.1)
2 (4.1)
3 (6.1)
17 (34.7)
2
50
14 (28.0)
2 (4.0)
5 (10.0)
29 (48.0)
3
50
15 (30.0)
1 (2.0)
0
34 (68.0)
4
47
19 (40.4)
1 (2.1)
0
27 (57.5)
(%)
=A total of 196 (99.0%) symptom sheets were returned for 198 vaccine doses administered Table 2 Overall percentages of injections with or without symptoms following vaccination with the hepatitis A candidate vaccine
With symptoms" Vaccine
Local only (%)
General only (%)
Local and general (%)
Without
180
54 (30.0)
12 (6.7)
8 (4.4)
106 (58.9)
360
75 (38.3)
6 (3.1)
8 (4.1)
107 (54.5)
720
12 (37.5)
1 (3.1)
2 (6.3)
17 (53.1)
Total
141
19
18
230
dose
(EU)
symptoms
(%)
°A total of 408 (98.8%) symptom sheets were returned for 413 vaccine doses administered
shown in Table 2. No significant differences were observed between the percentage of subjects reporting symptoms following doses of either 180, 360 or 720 EU. Overall, 56% (230/408) of documented doses did not elicit symptoms. Approximately one third (141/408) of documented injections were followed by only local symptoms, essentially mild soreness at the site of injection which lasted for I-2 days (Table 3). The most frequently reported general symptom following vaccination was headache (26/408 documented injections). Non-solicited symptoms included mostly feelings of 'fatigue' or 'tiredness'. None were considered serious.
Evaluation of vaccine immunogenicity The immune response of subjects following vaccination was assessed using an ELISA method (Table 4). After the first vaccine dose, 22/44 (50%) of the vaccinees in the lowest dose group, 180EU, had measurable titres of anti-HAV antibodies whereas 94% (47/50) and 88% (7/8) had seroconverted in the groups given 360 and 720 EU, respectively. One month after the third vaccine dose, only one subject given 180EU had not seroconverted; the administration of a fourth dose to this subject at 1 year resulted in the appearance of anti-HAV. After each vaccine dose, the geometric mean anti-HAV titre (GMT) increased in all groups. The GMTs of the group of subjects receiving 360 EU was significantly higher (p ~
Keywords:Candidate vaccine; hepatitis A; ELISA; inactivated
Introduction
Subjects and methods
Hepatitis A is a c o m m o n infectious disease present throughout the world. It is more prevalent in areas of poor sanitary conditions and low socioeconomic levels although sporadic outbreaks have been shown to occur in developed countries 1. Male homosexuals and intravenous drug addicts have been shown to be at increased risk of infection 2'3. Rates of infection are high in institutions such as pre-school day-care centres, prisons and mental hospitals where large groups of people are present under crowded conditions 3-5. Travellers to developing countries and military personnel are also at-risk groups 6-a. Although, unlike hepatitis B, hepatitis A does not lead to chronic infection, it is nevertheless an important cause of morbidity. At present, the only means of specific prophylaxis is passive immunization with immunoglobulins (ISG). The protection afforded by ISG is, however, transient 9. For long-term protection of high-risk groups, a safe and effective vaccine would be the most cost beneficial. In 1979, Provost and Hilleman 1° showed that the hepatitis A virus could be maintained in culture, permitting the development of both live, attenuated 11-13 and inactivated 14-17 candidate vaccines. The results of a clinical study in humans with a formaldehydeinactivated, whole-virion vaccine are reported here.
Trial p r o t o c o l A group of 110 healthy volunteers without antibodies against the hepatitis A virus initially gave their consent to participate in this study. They were allocated into three groups, the first two being randomized. Three subjects were found to have been infected with hepatitis B and were therefore excluded from the study. Two volunteers received vaccine at 0, 2 and 3 months, one other failed to return after one dose of vaccine; these subjects are not included in the analysis. The remaining volunteers were aged between 17 and 55 years (average of 26.7 years). The ratio of males to females was 0.93. The vaccine was administered in the deltoid muscle following a 0, 1, 2 month vaccination schedule. An additional dose of vaccine was given at month 12. Each dose contained either 180, 360 or 720 ELISA units (EU, see below) of killed hepatitis A virus. Blood samples for antibody determination were drawn before, and 1 month after, each vaccine.dose. In order to compare the immune response elicited by the candidate vaccine with anti-HAV titres following passive immunization, 23 seronegative volunteers (mean age 23.1 years; ratio of males to females, 0.83) were given 100IU per 60kg body weight of ISG (Globuman Hepatitis A Berna, Swiss Serum and Vaccine Institute). Serum samples were obtained 5 days later and anti-HAV titres evaluated by ELISA. Vaccine safety and reactogenicity were assessed by means of standardized checklists, where local, general or 'other' signs and symptoms were recorded on a four point, absent to severe, scale on the day of vaccination and for 3 days after each vaccine dose. Serum levels of liver-specific enzymes (aspartate aminotransferase, alanine aminotransferase, y-glutamyltranspeptidase and alkaline phosphatase) and serum bilirubin concentrations were also measured before and after vaccination.
*Institute of Specific Prophylaxis and Tropical Medicine, University of Vienna, Austria. t Institute of Virology, University of Vienna, Austria. tSmithKline Biologicals, Rixensart, Belgium. §To whom correspondence should be addressed at: Institute for Spezifische Prophylaxe und Tropenmedizin der Universit&t, Kinderspitalgasse 15, 1095 Vienna, Austria. (Received 30 April 1990; accepted 11 June 1990) 0264-410X/90/060581---04 © 1990 Butterworth-HeinernannLtd
Vaccine, Vol. 8, December 1990 581
Inactivated hepatitis A candidate vaccine: G. Wiedermann et al.
The study was conducted in accordance with the provisions of the Declaration of Helsinki. The trial was approved by the ethical commission of the University of Vienna. Written informed consent was obtained from a l l volunteers before vaccination.
Table 1 Percentages of vaccinees with or without symptoms following vaccination with 360 EU of the hepatitis A candidate vaccine Subjects with symptoms
Subjects Vaccine
Number of
dose
documented
Vaccine
number
injections"
The vaccine was prepared from the C L F strain (RIT 4379) grown on MRC-5 cells. The virus was purified by ultrafiltration and chromatography and inactivated with formaldehyde, as described previously 17. The vaccine meets the requirements of the W H O and the US Food and Drug Administration for similar inactivated vaccines~ 8,~9. The antigen content of the vaccine was measured by means of a capture ELISA (using purified peroxidase labelled IgG conjugate, see Ref. 17). Three different doses with 180, 360 and 720EU, absorbed onto 0.5mg of AI(OH)3 were prepared.
1
Serology Liver enzyme activities and serum bilirubin levels were measured using standard laboratory techniques. Antibodies against HAV were measured using an ELISA inhibition assay. Titres were calculated in mlU ml-1 in comparison with a standardized immunoglobulin preparation (obtained from the WHO) using the four parameter method 2°. Sera with titres below 20 mlU ml-1 were considered negative. Anti-HAV were also quantified using a commercially available radioimmunoassay (RIA; HAVAB, Abbott Laboratories, North Chicago, IL) and expressed in percentage inhibition. In accordance with the manufacturer's instructions, only those sera which inhibited binding by more than 50% were taken as positive. Neutralizing antibodies were assayed using the radioimmunofocus inhibition test (RIFIT, Ref. 21) on MRC-5 cells, using serial dilutions of serum (starting at 1/40 or 1/80 as indicated). RIFIT titres are expressed as the reciprocal of the highest serum dilution neutralizing 50% of the HAV foci. Purified virus.was prepared as described previously 22.
Statist&al analysis z 2 analysis and Student's t test, as indicated, were used to compare results. Statistical comparisons on the immune response in subjects administered 720 EU were not performed as this group was vaccinated separately at the end of the study.
Results Evaluation of vaccine safety and reactogenlcity Liver function tests to evaluate any eventual vaccineinduced hepatocellular damage were performed on serum samples from volunteers throughout the vaccination course. No clinically significant long-term increases in the activities of the four enzymes tested nor serum bilirubin concentrations were evidenced. In general, the frequency of adverse reactions decreased with successive vaccine doses, indicating that the candidate vaccine did not induce hypersensitivity reactions even after the administration of four doses. The results for volunteers given 360 EU doses are shown in
Table I. The effect of dose level on vaccine reactogenicity is
582
Vaccine, Vol. 8, D e c e m b e r 1990
Local only (%)
General only (%)
Local and general (%)
without symptoms
49
27 (55.1)
2 (4.1)
3 (6.1)
17 (34.7)
2
50
14 (28.0)
2 (4.0)
5 (10.0)
29 (48.0)
3
50
15 (30.0)
1 (2.0)
0
34 (68.0)
4
47
19 (40.4)
1 (2.1)
0
27 (57.5)
(%)
=A total of 196 (99.0%) symptom sheets were returned for 198 vaccine doses administered Table 2 Overall percentages of injections with or without symptoms following vaccination with the hepatitis A candidate vaccine
With symptoms" Vaccine
Local only (%)
General only (%)
Local and general (%)
Without
180
54 (30.0)
12 (6.7)
8 (4.4)
106 (58.9)
360
75 (38.3)
6 (3.1)
8 (4.1)
107 (54.5)
720
12 (37.5)
1 (3.1)
2 (6.3)
17 (53.1)
Total
141
19
18
230
dose
(EU)
symptoms
(%)
°A total of 408 (98.8%) symptom sheets were returned for 413 vaccine doses administered
shown in Table 2. No significant differences were observed between the percentage of subjects reporting symptoms following doses of either 180, 360 or 720 EU. Overall, 56% (230/408) of documented doses did not elicit symptoms. Approximately one third (141/408) of documented injections were followed by only local symptoms, essentially mild soreness at the site of injection which lasted for I-2 days (Table 3). The most frequently reported general symptom following vaccination was headache (26/408 documented injections). Non-solicited symptoms included mostly feelings of 'fatigue' or 'tiredness'. None were considered serious.
Evaluation of vaccine immunogenicity The immune response of subjects following vaccination was assessed using an ELISA method (Table 4). After the first vaccine dose, 22/44 (50%) of the vaccinees in the lowest dose group, 180EU, had measurable titres of anti-HAV antibodies whereas 94% (47/50) and 88% (7/8) had seroconverted in the groups given 360 and 720 EU, respectively. One month after the third vaccine dose, only one subject given 180EU had not seroconverted; the administration of a fourth dose to this subject at 1 year resulted in the appearance of anti-HAV. After each vaccine dose, the geometric mean anti-HAV titre (GMT) increased in all groups. The GMTs of the group of subjects receiving 360 EU was significantly higher (p ~
