Effect of virus strain and antigen dose on immunogenicity and reactogenicity of an inactivated hepatitis A vaccine Patrick Goubau*, Veerle Van Gerven t, Assad Safary;, Andr6e Delem ++, Jacqueline Knops t, Erik D'Hondt ~+,Francis E. Andr6 *+and Jan Desmyter*.~ A randomized double-blind comparison o f five killed hepatitis A vaccine preparations was carried out with eligible medical student and staff volunteers. Vaccines were prepared in M R C - 5 cells and formalin-inactivated. Three monthly injections o f 1 ml in the deltoid muscle were given. Group A received the C L F strain at a dose o f 360 E L I S A units (El. U) in 0.5 mg aluminium hydroxide (n = 35). The other groups received the HM175 strain as follows." 180 El. U in 1 mg aluminium hydroxide (to group B, n = 42), 360 El. U in 0.5 mg aluminium hydroxide (to group C, n = 40), 360 El.U in 1 mg aluminium hydroxide (to group D, n = 39) and 720 El. U in 1 mg aluminium hydroxide (to group E, n = 43). The geometric mean anti-HA V concentration ( G M C ) measured in m l V / m l by an E L I S A method one month after each injection were." group A, 223, 480, 1635; group B, 123, 221, 649," group C, 185,365, 1085; group D, 144, 323, 1076; group E, 229, 646, 2521. At month 6, the G M C had fallen by ~ 20%. Seroconversion as measured by E L I S A was 100% in groups A and E after one injection, and 100% in all groups after three injections," after two injections, only bne subject in group C was still negative. The dose effect with HM175 vaccine was significant. There was a good correlation between E L I S A and neutralization (radioimmunofocus inhibition test) titres. One month after the second dose, all subjects in groups A and E had both hepatitis A virus immunoglobulin M ( H A V IgM) geometric mean titre, ( G M T > 5000) and IgG ( G M T > 25 000) as measured by a sensitive terminal dilution E L I S A . All subjects in groups A and E became positive in the Abbott total anti-HA V radioimmunoassav, although at a titre lower than that after natural infection. Vaccinees in this stud), were negative in the Abbott I g M radioimmunoassay. Side effects were frequent but negligible, and essentially attributable to the aluminium adjuvant. Based on these results, and because the yield in cell culture o f the HM175 strain was better than that of the C L F strain, a 720 El. U dose o f HM175 was chosen as the standard vaccine formulation. In view o f the exceptionally powerful response after a single dose o f this vaccine (seroconversion 100% in this study and almost 100% in combined studies) two injections as a primary immunization course, plus a booster, give a satL~[actory response. Keywords: Virus strain; dose effect; anti-HAV response
INTRODUCTION Hepatitis A virus (HAV) infection often results in debilitating disease, particularly, in adults, and can sometimes be fatal. A l t h o u g h the incipience o f hepatitis A is declin*Department of Microbiology, Rega Institute and University Hospitals, Minderbroedersstraat, 10, B-3000 Leuven, Belgium. tDepartment of Youth Health Care, School of Public Health, Katholieke Universiteit Leuven, Kapucijnenvoer, 35, B-3000 Leuven, Belgium. ~SmithKline Beecham Biologicals, rue de I'lnstitut, 89, B-1330 Rixensart, Belgium. §To whom correspondence should be addressed
S114
Vaccine, Vol. 10, Suppl. 1, 1992
ing in m a n y industrialized countries, this contagious disease is still a worldwide problem and outbreaks occur sporadically, irrespective of geographical location t 3. Travellers from areas of low incidence to areas with a high incidence o f hepatitis A are particularly at risk. In industrialized countries, outbreaks are sometimes centred on day-care centres, where carers and parents contract the infection from children who are themselves often asymptomatic 4. The need for a vaccine has been evident for some time, but it was not until it became possible to adapt the virus to in vitro culture 5 that the development of a vaccine 0264-410X/92/100S114-05 © 1992Butterworth-HeinemannLtd
Virus strain and antigen dose in inactivated hepatitis A vaccine: P. Goubau et al.
became feasible. An inactivated candidate vaccine using the CLF strain was first developed, which was found to be safe and immunogenic in adults and in children. Later, a similar type of vaccine containing the HMI75 strain, which has a higher yield in cell culture than the CLF strain, was prepared. The present study was one of the first to be undertaken with the two vaccine candidates. It was intended to determine the feasibility of using the HMI75 strain in place of the CLF strain in the final vaccine. The study also evaluated different dose levels of the HM175 antigen and two different concentrations of aluminium hydroxide as adjuvant.
MATERIALS AND METHODS
Study participants A total of 203 subjects, 187 medical students and 16 staff members of the University Hospital, between the ages of 18 and 30 years, were enrolled and analysable (Table 1). Volunteers were in good health at entry and women were advised not to become pregnant during the study. Subjects were excluded from the study if they had travelled to a country of high HAV endemicity within the previous three months, had a history of liver disease or chronic alcohol consumption, were seropositive for antiHAV antibodies or had hepatitis B surface antigen, or had elevated serum liver enzyme activity (alanine aminotransferase/aspartate aminotransferase, ALT/AST) at screening. Informed consent was obtained from all subjects prior to enrolment. The study was approved by the Ethical Review Committee of the Katholeke Universiteit Leuven and was conducted in accordance with the provisions of the Declaration of Helsinki and its amendments.
Vaccine The candidate inactivated hepatitis A vaccines used in this study were prepared by SmithKline Beecham Biologicals (Rixensart, Belgium) using classical cell culture methods. One vaccine had been developed using the CLF virus strain (RIT 4379), isolated by Siegl et al. 5 from human stool. The isolation of the H M I 7 5 strain (RIT 4380) in primary monkey kidney cells from human stool and its subsequent culture was undertaken by Dr I. Gust (Australia) and Dr R. Purcell (USA). Both viral strains were propagated in MRC-5 cells. The viruses were purified by ultrafiltration and gel filtration, and inactivated with formaldehyde 6. Five vaccine formulations were prepared containing different antigen doses (expressed in
ELISA units, El.U) and with either 0.5 or 1 mg ofaluminium hydroxide per 1 ml dose (Table 1).
Study design Three doses of the candidate inactivated hepatitis A vaccine were administered intramuscularly in the deltoid region at one month intervals. A fourth vaccine dose, containing 720 E1.U of the HM175 formulation was administered to all subjects at month 12. Blood samples were taken at months 0, 1, 2, 3, 6, 12 and 13 for analysis of transaminases (ALT/AST) and for titration of antiHAV antibodies. A thorough physical examination was given and a medical history was taken at the first visit, with further evaluation at months 1,2 and 12. On the day of each vaccination and for the three following days, systemic reactions (headache, gastrointestinal symptoms, dizziness, fatigue, fever) and local reactions (soreness, induration, redness, swelling) and any other findings were recorded by the vaccinee on symptom sheets. The temperature was recorded (°C) and local redness and induration were measured (ram). Other systemic and local symptoms were to be scored similarly by the vaccinee as absent, mild, moderate or severe.
Laboratory analysis Serum liver enzyme activities (ALT/AST) were measured using standard laboratory techniques. Two techniques were used to assay vaccine-induced anti-HAV antibodies in serum: enzyme-linked immunosorbent assay (ELISA) and radioimmunofocus inhibition test (RIFIT) 7.~. Anti-HAV immunoglobulins M and G (IgM and IgG) were tested in three subgroups with a sensitive in-house ELISA, and IgM was tested with a commercial RIA (HAVAB-M, Abbott). Total antibodies against HAV were also measured using an ELISA inhibition assay, developed at SmithKline Beecham Biologicals, Belgium. Subjects with titres < 20 mIU/ml were considered to be seronegative. An increase in antibody titre from < 20mIU/ml to > 20 mIU/ml was considered to be a seroconversion.
Analysis of results X: analysis, analysis of variance (ANOVA) and Student's t test, as appropriate, were used to compare the results in each group. A geometric mean concentration (GMC) was calculated for the group at each time point for which samples were taken. The G M C of a population group is calculated as the antilog of the mean of the logs of the individual antibody concentrations. RESULTS
Table 1
Groups of vaccinees and their respective vaccine details Vaccine Aluminium No. of
hydroxide
Group
subjects
Strain
Dose (El.U)
(mg)
A B C D E
42 38 40 39 44
CLF HM175 HM175 HM175 HM175
360 180 360 360 720
0.5 1 0.5 1 1
A total of 203 initially anti-HAV negative subjects were enrolled in the study, aged 18 to 30 years (mean 20.2 + 1.86 years SD) and randomized into five groups. Analysis of variance, comparison of mean ages between groups and Student's t test comparing the means between males and females failed to show any significant differences between the groups.
Safety and reactogenicity of the vaccine Vaccine safety and reactogenicity were compared for the first three vaccine doses. The type and incidence of
Vaccine, Vol. 10, Suppl. 1, 1992
S115
Virus strain and antigen dose in inactivated hepatitis A vaccine." P. Goubau et al.
symptoms reported after the fourth vaccine dose (HM 175 strain. 720 E1.U for all subjects) were similar to those reported after the first three vaccinations. For these injections, a total of 589 symptom sheets were returned of 596 expected (99% compliance). The reactogenicity observed over the first three doses showed that the percentage of subjects reporting solicited or unsolicited symptoms decreased with each successive dose in most groups (Table 2) and no difference was observed in the frequency of symptoms between dose level or strain. However, for an equal quantity of antigen, the incidence of reactions was greater in the group with the higher concentration of aluminium (group D) than the group with the lower concentration (group C). Overall, 31% of documented injections were symptomfree. Symptoms were generally mild to moderate in severity and, if severe, were generally short-lasting (1 2 days), except for one subject who reported 4 consecutive days of severe fatigue after the third dose. The most common general symptom was fatigue, reported following 16% of documented injections, followed by headache (8.5%). Other general symptoms occurred with an incidence of < 5%. Soreness at the site of injection was reported following 51% of documented injections, with induration reported following 11%. Redness and swelling were reported after 4.2% and 2.9% of documented injections, respectively. Stiffness accounted for almost one-half of the unsolicited symptoms reported during the study. None of the symptoms was considered by subjects to be severe. Three serious adverse events were reported in the weeks or months following an injection (one subject reported aseptic meningitis, one reported juvenile diabetes and
Table 2
Incidence of symptoms after each vaccine dose and overall
Group
Subjects (%) with symptoms after dose 1
2
3
Overall Subjects with incidence of no symptoms symptoms (%)
(%) A B C D E
81 79 70 92 93
64 79 51 74 63
37 67 51 65 66
58 75 58 77 74
(42) (25) (42) (23) (26)
This table includes both solicited and unsolicited symptoms reported on symptom sheets. Total number of symptom sheets = 589
Table 3
one reported infectious mononucleosis), none of which were considered to be directly or indirectly related to vaccination.
Immunogenicity of the vaccine The results of analysis for anti-HAV antibodies is shown in Tahle 3. In all groups, seropositivity rates and geometric concentrations (GMC) increased with successive vaccine doses. After the first vaccine dose, 71.4 100% of subjects had seroconverted as indicated by ELISA, with G M C s wlrying from 123 mlU/ml (antigen dose of 180El.U) to 2 2 9 m l U / m l (antigen dose of 720El.U) between groups. One month after the third dose of vaccine, all subjects were seropositive for anti-HAV antibody and G M C s attained 650 to 2409 mlU/ml (lowest to highest dose levels respectively). A clear dose-effect was observed at each blood sampling (groups B, D and E: ANOVA: p < 0.001) with the 720 E1.U dose inducing a significantly higher response after three doses than the 180 and 360 E1.U dose levels. Student's t test failed to establish any difference either between the two strains tested (CLF group A and HM175 group C, both at a dose of 360 El.U) or between the lot adsorbed on two different concentrations of ahiminium (groups C and D, both at a dose of 360 El.U). By month 6, the G M C s had fallen by ~ 20%. However, all subjects were still seropositive at month 12; G M C s ranged from 286 mlU/ml for group B (180 El.U) to 782 520 and 487 mIU/ml for groups A. C and D (360 El.U) and 1233 mIU/ml for group E (720 El.U). The booster dose tit month 12 evoked a greater than eight-fold increase in antibody concentrations in each group. The antibody response in each group was analysed according to gender one month after the third dose (fahle 4). The significant effect of dose was seen for males and females separately. In all groups, females had higher immune response than males. For certain groups, such differences were statistically significant (Student's t test). All subjects were aged between 18 and 30 years and therefore no analysis of their age-related response was undertaken. Weight as a contributing factor in the observation was not investigated. Neutralizing antibodies in two of the groups were measured using R I F I T (Table 5) and a good correlation was found between these restllts and those measured by ELISA. One month after the second vaccine dose > 90% of the samples tested were shown to neutralize HAV, indicating that the wlccine induces antibodies of this type.
Antibody in healthy adults after vaccination with inactivated hepatitis A vaccine Antibody as measured by ELISA at month 1
2
6
12
13
Group
n
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
A B C D E
3,3-35 41-42 37-40 36-37 42-43
100 71 93 85 100
223 123 185 144 229
100 100 97 100 100
480 221 365 323 646
100 100 100 100 100
1593 650 1085 1043 2409
100 100 100 100 100
1470 481 882 856 1822
100 100 100 100 100
782 286 520 487 1233
100 100 100 100 100
SR, Seropositivity rate; GMC, geometric mean concentration
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Vaccine, Vol. 10, Suppl. 1, 1992
GMC (mlU/ml) 7074 5004 6117 8106 14 598
Virus strain and antigen dose in inactivated hepatitis A vaccine: P. Goubau et al.
serum liver enzyme activities were observed after any vaccine dose. The immunogenicity of the H M l 7 5 strain was comparable to that of the C L F strain at a similar antigen concentration (360 El.U). Because the yield in cell culture of the HM 175 strain is better than that of the C L F strain, the H M 175 strain is more suitable for the production of commercial vaccine. The antibody response was dose-dependent; 720 E1.U provoked significantly higher antibody responses than the dose of 180 EI.U. The higher levels of antibody persisted to month 12. All subjects given the highest concentration of H M I 7 5 vaccine seroconverted after the first dose, while two or three doses were necessary for the lower concentrations. Almost all subjects had measurable titres of neutralizing anti-HAV antibodies after the second dose of vaccine. The third dose at month 2 therefore seems unnecessary for routine vaccination. The vaccine elicited an IgM response in most individuals, although at a lower level than in natural HAV infection as it was undetectable with a routine diagnostic test. It may thus be possible to distinguish a vaccineinduced antibody response from that resulting from a natural infection. Increasing the aluminium content of the vaccine did not induce a greater antibody response. The antibody response in groups receiving the 360 EI.U dose adsorbed on either 1 or 0.5 mg aluminium hydroxide was similar. As the lower concentration of aluminium was less reactogenic, it has been recommended for the final vaccine. In conclusion, based partly on these results and partly because the yield in cell culture of the HM 175 strain was better than that of the C L F strain, a 720 EI.U dose of H M I 7 5 was chosen as the standard vaccine formulation. In view of the exceptionally powerful response (100% seroconversion) to a single dose of this vaccine, two injections plus a booster are considered sufficient.
]'able 4 Anti-HAV according to gender one month after the third HAV vaccine dose Anti-HAV as measured by ELISA at month 3 Male
Female
Seroconversion
Seroconversion
Group
Rate
%
GMC (mlU/ ml)
Rate
(%)
GMC (mlU/ ml)
A B C D E
13/13 21/21 16/16 18/18 17/17
100 100 100 100 100
480 1029 785 692 1869
22/22 21/21 22/22 20/20 26/26
100 100 100 100 100
877 1875 1372 1508 2844
Table 5 Neutralizing antibody response in healthy adults after immunization with inactivated hepatitis A vaccine Response at Dose (El.U)
Month 1
360 720
Month 2
Rate
%
Rate
%
ND 7/17
ND 41
50/55 17/18
91 94
Neutralizing antibodies were measured by RIFIT; dilutions > 1:40 were considered positive ND, not determined
Sixty vaccinees were tested for anti-HAV IgM and IgG (Table 6). IgM anti-HAV was detectable with the sensitive ELISA in all vaccinees in the higher dosage groups, but was undetectable with the commercial RIA. The anti-HAV IgG appeared later than anti-HAV IgM, particularly with lower dosage of 180 E1.U.
REFERENCES DISCUSSION
1 Speirs, J. and Bidawid, S. Food and waterborne viral outbreaks in Canada and the United States. Can. Dis. Week. Rep. 1991,17, 113-114 2 Lowry, P.W., Levine, R., Stroup, D.F., Gunn, R.A., Wilder, M.H. and Konigsberg, C. Hepatitis A outbreak on a floating restaurant in Florida, 1986 Am. J. EpidemioL 1989, 129, 155-164 3 Wang, J., Hu, S., Liu, H., Hong, Y., Cao, S. and Wu, L. Risk factor analysis of an epidemic of hepatitis A in a factory in Shanghai Int. J. Epidemiol. 1990, 19, 435-458 4 Hadler, S.C., Wabster, H.M., Erben, J.J., Swanson, J.E. and Maynard, Y.E. Hepatitis A in day-care centres. N. Eng. J. Med. 1980, 38"2, 12221227 5 Siegl, G., de Chastonay, J. and Kronauer, G. Propagation and assay
The candidate inactivated hepatitis A vaccines used in this trial were well tolerated and immunogenic. Symptoms reported were mild to moderate in severity, and transient. The reactogenicity typically decreased with each vaccine dose and was not greater for the higher vaccine dose levels compared with the lower dose levels. Vaccine reactogenicity appeared to be linked to the concentration of adjuvant, rather than the quantity of antigen in the vaccine. No clinically significant changes in Table 6
HAV-specific Ig subclass responses in healthy adults after administration of inactivated hepatitis A vaccine Anti-HAV response at
Vaccine dose (EU)
Month 1
Month 2
IgM
180 360 720
IgG
IgM
IgG
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
16/20 (80) 20/20 (100) 20/20 (100)
> 2794 2020 9734
2•20 (10) 6/20 (30) 14/20 (70)
39 310 67 081
17/20(85) 20/20(100) 20/20(100)
1864 1355 7638
14/20 (70) 17/20 (85) 20/20 (100)
142 298 213 010 493 989
Response measured by limit dilution titres using in-house ELISA. SR, Seroconversion rate: number seroconverted/number tested with percentage in parentheses. GMC, Geometric mean concentration of limit dilutions by ELISA
Vaccine, Vol. 10, Suppl. 1, 1992
$117
Virus strain a n d antigen dose in inactivated hepatitis A vaccine." P. Goubau et al.
6 7
of hepatitis A virus in vitro. J. Virol. Methods 1984, 9, 53-67 Andre, F.E.. Hepburn. A. and D'Hondt, E. Inactivated candidate vaccines for hepatitis A. Prog Med. Virol. 1990, 37. 72-95 Lemon, S.M.. Binn, L.N. and Marchwicki, RH. Radioimmunofocus
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V a c c i n e , Vol. 10, Suppl. 1, 1992
assay for quantitation of hepatitis A virus m cell cultures J. Clin. MicrobioZ 1983, 17, 834-839 Lemon, S.M. and Binn, L.N. Serum neutralizing antibody response to hepatitis A virus. J. Infect. Dis. 1983, 148, 1033-1049
INTRODUCTION Hepatitis A virus (HAV) infection often results in debilitating disease, particularly, in adults, and can sometimes be fatal. A l t h o u g h the incipience o f hepatitis A is declin*Department of Microbiology, Rega Institute and University Hospitals, Minderbroedersstraat, 10, B-3000 Leuven, Belgium. tDepartment of Youth Health Care, School of Public Health, Katholieke Universiteit Leuven, Kapucijnenvoer, 35, B-3000 Leuven, Belgium. ~SmithKline Beecham Biologicals, rue de I'lnstitut, 89, B-1330 Rixensart, Belgium. §To whom correspondence should be addressed
S114
Vaccine, Vol. 10, Suppl. 1, 1992
ing in m a n y industrialized countries, this contagious disease is still a worldwide problem and outbreaks occur sporadically, irrespective of geographical location t 3. Travellers from areas of low incidence to areas with a high incidence o f hepatitis A are particularly at risk. In industrialized countries, outbreaks are sometimes centred on day-care centres, where carers and parents contract the infection from children who are themselves often asymptomatic 4. The need for a vaccine has been evident for some time, but it was not until it became possible to adapt the virus to in vitro culture 5 that the development of a vaccine 0264-410X/92/100S114-05 © 1992Butterworth-HeinemannLtd
Virus strain and antigen dose in inactivated hepatitis A vaccine: P. Goubau et al.
became feasible. An inactivated candidate vaccine using the CLF strain was first developed, which was found to be safe and immunogenic in adults and in children. Later, a similar type of vaccine containing the HMI75 strain, which has a higher yield in cell culture than the CLF strain, was prepared. The present study was one of the first to be undertaken with the two vaccine candidates. It was intended to determine the feasibility of using the HMI75 strain in place of the CLF strain in the final vaccine. The study also evaluated different dose levels of the HM175 antigen and two different concentrations of aluminium hydroxide as adjuvant.
MATERIALS AND METHODS
Study participants A total of 203 subjects, 187 medical students and 16 staff members of the University Hospital, between the ages of 18 and 30 years, were enrolled and analysable (Table 1). Volunteers were in good health at entry and women were advised not to become pregnant during the study. Subjects were excluded from the study if they had travelled to a country of high HAV endemicity within the previous three months, had a history of liver disease or chronic alcohol consumption, were seropositive for antiHAV antibodies or had hepatitis B surface antigen, or had elevated serum liver enzyme activity (alanine aminotransferase/aspartate aminotransferase, ALT/AST) at screening. Informed consent was obtained from all subjects prior to enrolment. The study was approved by the Ethical Review Committee of the Katholeke Universiteit Leuven and was conducted in accordance with the provisions of the Declaration of Helsinki and its amendments.
Vaccine The candidate inactivated hepatitis A vaccines used in this study were prepared by SmithKline Beecham Biologicals (Rixensart, Belgium) using classical cell culture methods. One vaccine had been developed using the CLF virus strain (RIT 4379), isolated by Siegl et al. 5 from human stool. The isolation of the H M I 7 5 strain (RIT 4380) in primary monkey kidney cells from human stool and its subsequent culture was undertaken by Dr I. Gust (Australia) and Dr R. Purcell (USA). Both viral strains were propagated in MRC-5 cells. The viruses were purified by ultrafiltration and gel filtration, and inactivated with formaldehyde 6. Five vaccine formulations were prepared containing different antigen doses (expressed in
ELISA units, El.U) and with either 0.5 or 1 mg ofaluminium hydroxide per 1 ml dose (Table 1).
Study design Three doses of the candidate inactivated hepatitis A vaccine were administered intramuscularly in the deltoid region at one month intervals. A fourth vaccine dose, containing 720 E1.U of the HM175 formulation was administered to all subjects at month 12. Blood samples were taken at months 0, 1, 2, 3, 6, 12 and 13 for analysis of transaminases (ALT/AST) and for titration of antiHAV antibodies. A thorough physical examination was given and a medical history was taken at the first visit, with further evaluation at months 1,2 and 12. On the day of each vaccination and for the three following days, systemic reactions (headache, gastrointestinal symptoms, dizziness, fatigue, fever) and local reactions (soreness, induration, redness, swelling) and any other findings were recorded by the vaccinee on symptom sheets. The temperature was recorded (°C) and local redness and induration were measured (ram). Other systemic and local symptoms were to be scored similarly by the vaccinee as absent, mild, moderate or severe.
Laboratory analysis Serum liver enzyme activities (ALT/AST) were measured using standard laboratory techniques. Two techniques were used to assay vaccine-induced anti-HAV antibodies in serum: enzyme-linked immunosorbent assay (ELISA) and radioimmunofocus inhibition test (RIFIT) 7.~. Anti-HAV immunoglobulins M and G (IgM and IgG) were tested in three subgroups with a sensitive in-house ELISA, and IgM was tested with a commercial RIA (HAVAB-M, Abbott). Total antibodies against HAV were also measured using an ELISA inhibition assay, developed at SmithKline Beecham Biologicals, Belgium. Subjects with titres < 20 mIU/ml were considered to be seronegative. An increase in antibody titre from < 20mIU/ml to > 20 mIU/ml was considered to be a seroconversion.
Analysis of results X: analysis, analysis of variance (ANOVA) and Student's t test, as appropriate, were used to compare the results in each group. A geometric mean concentration (GMC) was calculated for the group at each time point for which samples were taken. The G M C of a population group is calculated as the antilog of the mean of the logs of the individual antibody concentrations. RESULTS
Table 1
Groups of vaccinees and their respective vaccine details Vaccine Aluminium No. of
hydroxide
Group
subjects
Strain
Dose (El.U)
(mg)
A B C D E
42 38 40 39 44
CLF HM175 HM175 HM175 HM175
360 180 360 360 720
0.5 1 0.5 1 1
A total of 203 initially anti-HAV negative subjects were enrolled in the study, aged 18 to 30 years (mean 20.2 + 1.86 years SD) and randomized into five groups. Analysis of variance, comparison of mean ages between groups and Student's t test comparing the means between males and females failed to show any significant differences between the groups.
Safety and reactogenicity of the vaccine Vaccine safety and reactogenicity were compared for the first three vaccine doses. The type and incidence of
Vaccine, Vol. 10, Suppl. 1, 1992
S115
Virus strain and antigen dose in inactivated hepatitis A vaccine." P. Goubau et al.
symptoms reported after the fourth vaccine dose (HM 175 strain. 720 E1.U for all subjects) were similar to those reported after the first three vaccinations. For these injections, a total of 589 symptom sheets were returned of 596 expected (99% compliance). The reactogenicity observed over the first three doses showed that the percentage of subjects reporting solicited or unsolicited symptoms decreased with each successive dose in most groups (Table 2) and no difference was observed in the frequency of symptoms between dose level or strain. However, for an equal quantity of antigen, the incidence of reactions was greater in the group with the higher concentration of aluminium (group D) than the group with the lower concentration (group C). Overall, 31% of documented injections were symptomfree. Symptoms were generally mild to moderate in severity and, if severe, were generally short-lasting (1 2 days), except for one subject who reported 4 consecutive days of severe fatigue after the third dose. The most common general symptom was fatigue, reported following 16% of documented injections, followed by headache (8.5%). Other general symptoms occurred with an incidence of < 5%. Soreness at the site of injection was reported following 51% of documented injections, with induration reported following 11%. Redness and swelling were reported after 4.2% and 2.9% of documented injections, respectively. Stiffness accounted for almost one-half of the unsolicited symptoms reported during the study. None of the symptoms was considered by subjects to be severe. Three serious adverse events were reported in the weeks or months following an injection (one subject reported aseptic meningitis, one reported juvenile diabetes and
Table 2
Incidence of symptoms after each vaccine dose and overall
Group
Subjects (%) with symptoms after dose 1
2
3
Overall Subjects with incidence of no symptoms symptoms (%)
(%) A B C D E
81 79 70 92 93
64 79 51 74 63
37 67 51 65 66
58 75 58 77 74
(42) (25) (42) (23) (26)
This table includes both solicited and unsolicited symptoms reported on symptom sheets. Total number of symptom sheets = 589
Table 3
one reported infectious mononucleosis), none of which were considered to be directly or indirectly related to vaccination.
Immunogenicity of the vaccine The results of analysis for anti-HAV antibodies is shown in Tahle 3. In all groups, seropositivity rates and geometric concentrations (GMC) increased with successive vaccine doses. After the first vaccine dose, 71.4 100% of subjects had seroconverted as indicated by ELISA, with G M C s wlrying from 123 mlU/ml (antigen dose of 180El.U) to 2 2 9 m l U / m l (antigen dose of 720El.U) between groups. One month after the third dose of vaccine, all subjects were seropositive for anti-HAV antibody and G M C s attained 650 to 2409 mlU/ml (lowest to highest dose levels respectively). A clear dose-effect was observed at each blood sampling (groups B, D and E: ANOVA: p < 0.001) with the 720 E1.U dose inducing a significantly higher response after three doses than the 180 and 360 E1.U dose levels. Student's t test failed to establish any difference either between the two strains tested (CLF group A and HM175 group C, both at a dose of 360 El.U) or between the lot adsorbed on two different concentrations of ahiminium (groups C and D, both at a dose of 360 El.U). By month 6, the G M C s had fallen by ~ 20%. However, all subjects were still seropositive at month 12; G M C s ranged from 286 mlU/ml for group B (180 El.U) to 782 520 and 487 mIU/ml for groups A. C and D (360 El.U) and 1233 mIU/ml for group E (720 El.U). The booster dose tit month 12 evoked a greater than eight-fold increase in antibody concentrations in each group. The antibody response in each group was analysed according to gender one month after the third dose (fahle 4). The significant effect of dose was seen for males and females separately. In all groups, females had higher immune response than males. For certain groups, such differences were statistically significant (Student's t test). All subjects were aged between 18 and 30 years and therefore no analysis of their age-related response was undertaken. Weight as a contributing factor in the observation was not investigated. Neutralizing antibodies in two of the groups were measured using R I F I T (Table 5) and a good correlation was found between these restllts and those measured by ELISA. One month after the second vaccine dose > 90% of the samples tested were shown to neutralize HAV, indicating that the wlccine induces antibodies of this type.
Antibody in healthy adults after vaccination with inactivated hepatitis A vaccine Antibody as measured by ELISA at month 1
2
6
12
13
Group
n
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
GMC (mlU/ml)
SR(%)
A B C D E
3,3-35 41-42 37-40 36-37 42-43
100 71 93 85 100
223 123 185 144 229
100 100 97 100 100
480 221 365 323 646
100 100 100 100 100
1593 650 1085 1043 2409
100 100 100 100 100
1470 481 882 856 1822
100 100 100 100 100
782 286 520 487 1233
100 100 100 100 100
SR, Seropositivity rate; GMC, geometric mean concentration
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GMC (mlU/ml) 7074 5004 6117 8106 14 598
Virus strain and antigen dose in inactivated hepatitis A vaccine: P. Goubau et al.
serum liver enzyme activities were observed after any vaccine dose. The immunogenicity of the H M l 7 5 strain was comparable to that of the C L F strain at a similar antigen concentration (360 El.U). Because the yield in cell culture of the HM 175 strain is better than that of the C L F strain, the H M 175 strain is more suitable for the production of commercial vaccine. The antibody response was dose-dependent; 720 E1.U provoked significantly higher antibody responses than the dose of 180 EI.U. The higher levels of antibody persisted to month 12. All subjects given the highest concentration of H M I 7 5 vaccine seroconverted after the first dose, while two or three doses were necessary for the lower concentrations. Almost all subjects had measurable titres of neutralizing anti-HAV antibodies after the second dose of vaccine. The third dose at month 2 therefore seems unnecessary for routine vaccination. The vaccine elicited an IgM response in most individuals, although at a lower level than in natural HAV infection as it was undetectable with a routine diagnostic test. It may thus be possible to distinguish a vaccineinduced antibody response from that resulting from a natural infection. Increasing the aluminium content of the vaccine did not induce a greater antibody response. The antibody response in groups receiving the 360 EI.U dose adsorbed on either 1 or 0.5 mg aluminium hydroxide was similar. As the lower concentration of aluminium was less reactogenic, it has been recommended for the final vaccine. In conclusion, based partly on these results and partly because the yield in cell culture of the HM 175 strain was better than that of the C L F strain, a 720 EI.U dose of H M I 7 5 was chosen as the standard vaccine formulation. In view of the exceptionally powerful response (100% seroconversion) to a single dose of this vaccine, two injections plus a booster are considered sufficient.
]'able 4 Anti-HAV according to gender one month after the third HAV vaccine dose Anti-HAV as measured by ELISA at month 3 Male
Female
Seroconversion
Seroconversion
Group
Rate
%
GMC (mlU/ ml)
Rate
(%)
GMC (mlU/ ml)
A B C D E
13/13 21/21 16/16 18/18 17/17
100 100 100 100 100
480 1029 785 692 1869
22/22 21/21 22/22 20/20 26/26
100 100 100 100 100
877 1875 1372 1508 2844
Table 5 Neutralizing antibody response in healthy adults after immunization with inactivated hepatitis A vaccine Response at Dose (El.U)
Month 1
360 720
Month 2
Rate
%
Rate
%
ND 7/17
ND 41
50/55 17/18
91 94
Neutralizing antibodies were measured by RIFIT; dilutions > 1:40 were considered positive ND, not determined
Sixty vaccinees were tested for anti-HAV IgM and IgG (Table 6). IgM anti-HAV was detectable with the sensitive ELISA in all vaccinees in the higher dosage groups, but was undetectable with the commercial RIA. The anti-HAV IgG appeared later than anti-HAV IgM, particularly with lower dosage of 180 E1.U.
REFERENCES DISCUSSION
1 Speirs, J. and Bidawid, S. Food and waterborne viral outbreaks in Canada and the United States. Can. Dis. Week. Rep. 1991,17, 113-114 2 Lowry, P.W., Levine, R., Stroup, D.F., Gunn, R.A., Wilder, M.H. and Konigsberg, C. Hepatitis A outbreak on a floating restaurant in Florida, 1986 Am. J. EpidemioL 1989, 129, 155-164 3 Wang, J., Hu, S., Liu, H., Hong, Y., Cao, S. and Wu, L. Risk factor analysis of an epidemic of hepatitis A in a factory in Shanghai Int. J. Epidemiol. 1990, 19, 435-458 4 Hadler, S.C., Wabster, H.M., Erben, J.J., Swanson, J.E. and Maynard, Y.E. Hepatitis A in day-care centres. N. Eng. J. Med. 1980, 38"2, 12221227 5 Siegl, G., de Chastonay, J. and Kronauer, G. Propagation and assay
The candidate inactivated hepatitis A vaccines used in this trial were well tolerated and immunogenic. Symptoms reported were mild to moderate in severity, and transient. The reactogenicity typically decreased with each vaccine dose and was not greater for the higher vaccine dose levels compared with the lower dose levels. Vaccine reactogenicity appeared to be linked to the concentration of adjuvant, rather than the quantity of antigen in the vaccine. No clinically significant changes in Table 6
HAV-specific Ig subclass responses in healthy adults after administration of inactivated hepatitis A vaccine Anti-HAV response at
Vaccine dose (EU)
Month 1
Month 2
IgM
180 360 720
IgG
IgM
IgG
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
SR (%)
GMC (mlU/ml)
16/20 (80) 20/20 (100) 20/20 (100)
> 2794 2020 9734
2•20 (10) 6/20 (30) 14/20 (70)
39 310 67 081
17/20(85) 20/20(100) 20/20(100)
1864 1355 7638
14/20 (70) 17/20 (85) 20/20 (100)
142 298 213 010 493 989
Response measured by limit dilution titres using in-house ELISA. SR, Seroconversion rate: number seroconverted/number tested with percentage in parentheses. GMC, Geometric mean concentration of limit dilutions by ELISA
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Virus strain a n d antigen dose in inactivated hepatitis A vaccine." P. Goubau et al.
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of hepatitis A virus in vitro. J. Virol. Methods 1984, 9, 53-67 Andre, F.E.. Hepburn. A. and D'Hondt, E. Inactivated candidate vaccines for hepatitis A. Prog Med. Virol. 1990, 37. 72-95 Lemon, S.M.. Binn, L.N. and Marchwicki, RH. Radioimmunofocus
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assay for quantitation of hepatitis A virus m cell cultures J. Clin. MicrobioZ 1983, 17, 834-839 Lemon, S.M. and Binn, L.N. Serum neutralizing antibody response to hepatitis A virus. J. Infect. Dis. 1983, 148, 1033-1049
