Short Paper Immunogenicity trial of inactivated hepatitis A virus vaccine in human volunteers Yu.Yu. Kusov*, L.B. Elbert, I.V. Nelga, G.K. Grishina, O.A. Dunaevski, N.V. Kharin, Yu.N. Maslov, S.G. Drozdov and M.S. Balayan An inactivated hepatitis A virus (HA V) vacc&e was tested on a group o f human adult volunteers. The vaccine was administered subcutaneously, and a control group received a placebo (aluminium hydroxide). The vaccine was found to be relatively well tolerated and non-reactoaenic, and levels o f anti-HA V were comparable to those in other studies. Keywords: Hepatitis A; subcutaneous; placebo; aluminium hydroxide; human volunteers; inactivated
Some characteristics of hepatitis A virus (HAV) vaccine obtained in assays of immunogenicity and safety in laboratory animals, including marmoset monkeys susceptibleto hepatitis A infection, were previously reported1. The vaccine was shown to be safe, sufficiently immunogenic and capable of producing specific resistance to challenge inoculation with virulent HAV. A trial of this vaccine in two limited groups of human volunteers is now reported. The vaccine was prepared as previously described z. Briefly, HAV was grown in a Vero-like heteroploid cell line (adult vervet monkey kidney) licensed by national control authorities for production of inactivated virus vaccines for human use3; the virus was further purified by chloroform extraction followed by DNase treatment and column chromatography on modified porous silica beads, and inactivated by exposure to 0.02% formaldehyde. The vaccine (4-8 HAV antigen units per dose) was mixed with aluminium hydroxide (final concentration 1.1-1.3mg m1-1) just before administration. Laboratory control tests showed that the vaccine composition was in accordance with WHO requirements for inactivated virus vaccines produced in heteroploid cells4. The laboratory vaccine lot prepared for the trial and the trial design were approved by national control authorities. The study was conducted following the published ethical regulations for biomedical research involving human subjects s. Altogether 55 healthy individuals of both sexes aged between 18 and 56 years were serologically screened and selected for the study. Group 1 consisted of 15 laboratory staff members, of whom nine, including two seronegatives, were vaccinated and six received placebo preparation (aluminium hydroxide suspension). Group 2 comprised
Institute of Poliomyelitis and Viral Encephalitides, Moscow State Medical Institute, Tver, 142782 Moscow, USSR. *To whom correspondence should be addressed. (Received 6 September 1990; revised 31 January 1991; accepted
31 January 1991) ~10X/91/080540~)2 © 1991 Butterworth-HeinemannLtd
540 Vaccine, Vol. 9, August 1991
40 medical students, 17 of whom, all seronegative, were given the vaccine and 23 (nine seronegatives and 14 seropositives) the placebo. Aluminium-adsorbed vaccine or placebo preparation (1 ml)was injected subcutaneously at a level of the low angle of the scapula three times 4 weeks apart. All the individuals involved in the study passed thorough clinical examination including determination of serum alanine-aminotransferase (ALT) levels before the first injection. Twice-weekly examinations were continued until week 3 after the last injection. A special questionnaire for recording the individual complaints and clinically determined symptoms was used. At the end of the trial the completed questionnaires from all subjects were analysed. Blood samples were taken before each injection and 3 weeks after the last one. Anti-HAV antibody was measured by the blocking variant of enzyme-immunoassay6. Table 1 shows the occurrence of adverse reactions associated with vaccination. Burning at the injection site was the most common reaction, reported by almost every vaccinee after each injection. The duration of burning was 40-90 s immediately after the injection and may have been caused by the residual formaldehyde in the vaccine (0.005-0.01%). Fever was observed in three and local redness in one of the vaccinees 1 day after the first vaccination. Otherwise the HAV vaccine appeared to be well tolerated and non-reactogenic. ALT values remained within normal limits in all the vaccinees during the period of observation. Quantitative anti-HAV responses are shown in Figure I. The proportion of positive, i.e. seroconverted, individuals increased with successive doses though the range of antibody titres remained rather wide at each time interval. In addition, five individuals who had had anti-HAV at a level of 1:1000 to 1:6000 prior to vaccination responded to three vaccine doses by further antibody titre rise reaching levels of 1 :20 000 and above. There were no significant changes in the anti-HAV titres in individuals who received the placebo throughout the period of observation.
I m m u n o g e n i c i t y of i n a c t i v a t e d H A V vaccine: Yu.Yu. K u s o v et al. Table 1 Occurrence of adverse reactions after HAV vaccine and placebo HAV vaccine
Fever ~ 37.5°C General symptoms Headache Malaise Joint pain Anorexia Specific symptoms Liver enlargement Epigastric pain ALT elevation Local reactions Redness Swelling Protracted pain ~ Lymphadenitis
IO00 8O0 70C 60C 500
Placebo
Group 1 (n=9)
Group 2 (n=17)
Groups 1 + 2 (n=29)
1 (11.1%) a 0
2 (11.7%) 0
0 0
0 0 0 0
0 0 0 0
0 0 0 0
== E ~> "ri
0 0 0
0 0 0
0 0 0
g ~ ~
Ia 0 0 0
0 0 0 0
0 0 0 0
I
O
200
CONCLUSIONS This trial of inactivated HAV vaccine in humans indicates that the vaccine is relatively well tolerated and nonreactogenic. Three doses of the vaccine generated anti-HAV responses in all susceptible individuals and produced a booster effect in persons having moderate anti-HAV titres prior to vaccination. The levels of anti-HAV obtained by immunization of seronegatives are comparable with those demonstrated in other studies 7-1°, but they were lower than the anti-HAV titres acquired after natural hepatitis A infection 11. Whether or not the anti-HAV levels attained in vaccination are sufficient to prevent hepatitis A, and for h o w long, remains to be determined in a larger field trial.
REFERENCES
3
O
•
500
_J
IO0 90 80 70 60 50 40
°O
30 20
0
2
3
4OO
aFever and local redness were observed in one vaccinee after first vaccination ~Short-lasting burning on the injection site has been reported by many vaccinees (see text)
1
2
Kusov, Yu.Yu., Kazachkov, Yu.A., Elbert, L.B., Krutyanskaya, G.L., Poleschuk, V.F., Sobol, A.V. et al. Characteristics of inactivated hepatitis A vaccine prepared from virus propagated in heteroploid continuous monkey cell line. Vaccine 1990, 8, 513-514 Balayan, M.S. Development of hepatitis A virus vaccines. Vopr. Virusol. 1988, 1, 5-11 (in Russian) Mironova, E L , Shalunova, N.V., Lomanova, G.A., Nikolaeva, M.A., Khapchaev, Yu.H., Stobetsky, V.I. et al. Characteristics of the bank of 4647 heteroploid cells from vervet monkey kidney. Vopr. Virusol. 1987, 6, 740-743 (in Russian)
4 8 Time after Ist vaccination (weeks)
12
Figure 1 Anti-HAY response to three doses of vaccine in 19 individuals lacking this antibody before vaccination. Time after first vaccination is plotted against reciprocal values of anti-HAV-titres, measured by ELISA (block-version), in individual sera. Arrows indicate the time of vaccine injection. O, group 1 of volunteers; O, group 2 4
WHO Study Group. Acceptability of cell substrates for production of biologicals. WHO Technical Report Series 474 WHO, Geneva, 1987 5 Gutteridge, F. Human experimentation and medical ethics. International guidelines for biomedical research involving human subjects. WHO Chron. 1981, 35, 212-215 6 Duermeyer, W. Application of ELISA for Diagnosis and Epidemiology of Hepatitis A, Rodopi, Amsterdam, 1980 7 Flehmig, B., Heinricy, U. and Pfisterer, M. Immunogenicity of killed hepatitis A vaccine in seronegative volunteers. Lancet 1989, i, 1039-1041 8 Desmyter, J., Goubau, P., D'Hondt, E., Delem, A., Safary, A. and Andre, F. Immunogenicity and reactogenicity of five formulations of inactivated hepatitis A vaccine. 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Texas, Abstract no. 41 9 Binn, L.N., Sjogren, M., Marchwicki, R.H., MacArthy, P., Hake, C. and Bancroft, W.H. Antibody response of volunteers to inactivated hepatitis A virus vaccines. 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Texas, Abstract no. 46 10 Flehmig, B., Heinricy, U. and Pfisterer, M. Prospects for a hepatitis A virus vaccine. Prog. Med. Viral. 1989, 37, 15-31 11 Frosner, G.G., Overby, LR., Flehmig, B., Gerth, H.-J., Haas, H., Decker, R.H. etal. Seroepidemiological investigation of patients and family contacts in an epidemic of hepatitis A. J. Med. Viral. 1977, 1, 163-173
Vaccine, Vol. 9, A u g u s t 1991
541
Some characteristics of hepatitis A virus (HAV) vaccine obtained in assays of immunogenicity and safety in laboratory animals, including marmoset monkeys susceptibleto hepatitis A infection, were previously reported1. The vaccine was shown to be safe, sufficiently immunogenic and capable of producing specific resistance to challenge inoculation with virulent HAV. A trial of this vaccine in two limited groups of human volunteers is now reported. The vaccine was prepared as previously described z. Briefly, HAV was grown in a Vero-like heteroploid cell line (adult vervet monkey kidney) licensed by national control authorities for production of inactivated virus vaccines for human use3; the virus was further purified by chloroform extraction followed by DNase treatment and column chromatography on modified porous silica beads, and inactivated by exposure to 0.02% formaldehyde. The vaccine (4-8 HAV antigen units per dose) was mixed with aluminium hydroxide (final concentration 1.1-1.3mg m1-1) just before administration. Laboratory control tests showed that the vaccine composition was in accordance with WHO requirements for inactivated virus vaccines produced in heteroploid cells4. The laboratory vaccine lot prepared for the trial and the trial design were approved by national control authorities. The study was conducted following the published ethical regulations for biomedical research involving human subjects s. Altogether 55 healthy individuals of both sexes aged between 18 and 56 years were serologically screened and selected for the study. Group 1 consisted of 15 laboratory staff members, of whom nine, including two seronegatives, were vaccinated and six received placebo preparation (aluminium hydroxide suspension). Group 2 comprised
Institute of Poliomyelitis and Viral Encephalitides, Moscow State Medical Institute, Tver, 142782 Moscow, USSR. *To whom correspondence should be addressed. (Received 6 September 1990; revised 31 January 1991; accepted
31 January 1991) ~10X/91/080540~)2 © 1991 Butterworth-HeinemannLtd
540 Vaccine, Vol. 9, August 1991
40 medical students, 17 of whom, all seronegative, were given the vaccine and 23 (nine seronegatives and 14 seropositives) the placebo. Aluminium-adsorbed vaccine or placebo preparation (1 ml)was injected subcutaneously at a level of the low angle of the scapula three times 4 weeks apart. All the individuals involved in the study passed thorough clinical examination including determination of serum alanine-aminotransferase (ALT) levels before the first injection. Twice-weekly examinations were continued until week 3 after the last injection. A special questionnaire for recording the individual complaints and clinically determined symptoms was used. At the end of the trial the completed questionnaires from all subjects were analysed. Blood samples were taken before each injection and 3 weeks after the last one. Anti-HAV antibody was measured by the blocking variant of enzyme-immunoassay6. Table 1 shows the occurrence of adverse reactions associated with vaccination. Burning at the injection site was the most common reaction, reported by almost every vaccinee after each injection. The duration of burning was 40-90 s immediately after the injection and may have been caused by the residual formaldehyde in the vaccine (0.005-0.01%). Fever was observed in three and local redness in one of the vaccinees 1 day after the first vaccination. Otherwise the HAV vaccine appeared to be well tolerated and non-reactogenic. ALT values remained within normal limits in all the vaccinees during the period of observation. Quantitative anti-HAV responses are shown in Figure I. The proportion of positive, i.e. seroconverted, individuals increased with successive doses though the range of antibody titres remained rather wide at each time interval. In addition, five individuals who had had anti-HAV at a level of 1:1000 to 1:6000 prior to vaccination responded to three vaccine doses by further antibody titre rise reaching levels of 1 :20 000 and above. There were no significant changes in the anti-HAV titres in individuals who received the placebo throughout the period of observation.
I m m u n o g e n i c i t y of i n a c t i v a t e d H A V vaccine: Yu.Yu. K u s o v et al. Table 1 Occurrence of adverse reactions after HAV vaccine and placebo HAV vaccine
Fever ~ 37.5°C General symptoms Headache Malaise Joint pain Anorexia Specific symptoms Liver enlargement Epigastric pain ALT elevation Local reactions Redness Swelling Protracted pain ~ Lymphadenitis
IO00 8O0 70C 60C 500
Placebo
Group 1 (n=9)
Group 2 (n=17)
Groups 1 + 2 (n=29)
1 (11.1%) a 0
2 (11.7%) 0
0 0
0 0 0 0
0 0 0 0
0 0 0 0
== E ~> "ri
0 0 0
0 0 0
0 0 0
g ~ ~
Ia 0 0 0
0 0 0 0
0 0 0 0
I
O
200
CONCLUSIONS This trial of inactivated HAV vaccine in humans indicates that the vaccine is relatively well tolerated and nonreactogenic. Three doses of the vaccine generated anti-HAV responses in all susceptible individuals and produced a booster effect in persons having moderate anti-HAV titres prior to vaccination. The levels of anti-HAV obtained by immunization of seronegatives are comparable with those demonstrated in other studies 7-1°, but they were lower than the anti-HAV titres acquired after natural hepatitis A infection 11. Whether or not the anti-HAV levels attained in vaccination are sufficient to prevent hepatitis A, and for h o w long, remains to be determined in a larger field trial.
REFERENCES
3
O
•
500
_J
IO0 90 80 70 60 50 40
°O
30 20
0
2
3
4OO
aFever and local redness were observed in one vaccinee after first vaccination ~Short-lasting burning on the injection site has been reported by many vaccinees (see text)
1
2
Kusov, Yu.Yu., Kazachkov, Yu.A., Elbert, L.B., Krutyanskaya, G.L., Poleschuk, V.F., Sobol, A.V. et al. Characteristics of inactivated hepatitis A vaccine prepared from virus propagated in heteroploid continuous monkey cell line. Vaccine 1990, 8, 513-514 Balayan, M.S. Development of hepatitis A virus vaccines. Vopr. Virusol. 1988, 1, 5-11 (in Russian) Mironova, E L , Shalunova, N.V., Lomanova, G.A., Nikolaeva, M.A., Khapchaev, Yu.H., Stobetsky, V.I. et al. Characteristics of the bank of 4647 heteroploid cells from vervet monkey kidney. Vopr. Virusol. 1987, 6, 740-743 (in Russian)
4 8 Time after Ist vaccination (weeks)
12
Figure 1 Anti-HAY response to three doses of vaccine in 19 individuals lacking this antibody before vaccination. Time after first vaccination is plotted against reciprocal values of anti-HAV-titres, measured by ELISA (block-version), in individual sera. Arrows indicate the time of vaccine injection. O, group 1 of volunteers; O, group 2 4
WHO Study Group. Acceptability of cell substrates for production of biologicals. WHO Technical Report Series 474 WHO, Geneva, 1987 5 Gutteridge, F. Human experimentation and medical ethics. International guidelines for biomedical research involving human subjects. WHO Chron. 1981, 35, 212-215 6 Duermeyer, W. Application of ELISA for Diagnosis and Epidemiology of Hepatitis A, Rodopi, Amsterdam, 1980 7 Flehmig, B., Heinricy, U. and Pfisterer, M. Immunogenicity of killed hepatitis A vaccine in seronegative volunteers. Lancet 1989, i, 1039-1041 8 Desmyter, J., Goubau, P., D'Hondt, E., Delem, A., Safary, A. and Andre, F. Immunogenicity and reactogenicity of five formulations of inactivated hepatitis A vaccine. 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Texas, Abstract no. 41 9 Binn, L.N., Sjogren, M., Marchwicki, R.H., MacArthy, P., Hake, C. and Bancroft, W.H. Antibody response of volunteers to inactivated hepatitis A virus vaccines. 1990 International Symposium on Viral Hepatitis and Liver Disease, Houston, Texas, Abstract no. 46 10 Flehmig, B., Heinricy, U. and Pfisterer, M. Prospects for a hepatitis A virus vaccine. Prog. Med. Viral. 1989, 37, 15-31 11 Frosner, G.G., Overby, LR., Flehmig, B., Gerth, H.-J., Haas, H., Decker, R.H. etal. Seroepidemiological investigation of patients and family contacts in an epidemic of hepatitis A. J. Med. Viral. 1977, 1, 163-173
Vaccine, Vol. 9, A u g u s t 1991
541
